Resistance to antibacterial agents was the subject of a microbiology symposium
held at the British Pharmaceutical Conference on September 11 and organised
in conjunction with the British Society of Antimicrobial Chemotherapy.
The symposium was divided into two parts: a historical overview and current
research; and future developments
Antibiotic resistance is nothing new
Chairing the first part of the microbiology symposium with Professor PAUL WILLIAMS
(institute of infections and
immunity and school of pharmaceutical
sciences, University of Nottingham), Professor RICHARD WISE, chairman of the
British Society of Antimicrobial Chemotherapy, said that there was nothing new
about antimicrobial resistance — it had been a problem since the first
antimicrobial agents came onto the market — and it was a simple process
of Darwinian selection pressure. The symposium was focusing only on resistance
to antibacterial agents, but the area of antimicrobial resistance was much broader,
and included, for example, disinfectants. Professor Wise emphasised that antimicrobial
resistance was “an extraordinarily important area . . . and one that governments
around the world are now trying to get to grips with”. In the United States,
the National Institutes of Health and the Centers for Disease Control and Prevention
had recently been granted $500m to tackle the problem. Unfortunately, sums of
money of this size were not being made available in the United Kingdom.
Historical overview
Professor HUGH PENNINGTON (department of medical microbiology, University of
Aberdeen) gave an overview of the problem of antimicrobial resistance. Setting
the scene from a historical perspective, Professor Pennington suggested that
perhaps the greatest advance in medicine of the 19th century was the introduction
of antiseptics and, later, aseptic surgery. The contribution that antibiotics
had made in medicine was astounding — sulphonamides, discovered in the
1930s, had had a revolutionary impact on sepsis at childbirth, and penicillin,
discovered in the 1920s, had played a crucial role in medicine during the 1939-45
war.
However, according to Professor Pennington, the organisms that caused problems
in the early 19th century were the same organisms that caused problems today,
from an antimicrobial point of view. It was in the late 1960s that problems
with antibiotic resistance began to occur. Talking about the role of evolution
in the development of antimicrobial resistance, Professor Pennington explained
that bacteria, viruses and microbes in general evolved well as they had short
generation times.
“In any branch of medicine, we forget about evolution at our peril,”
he warned. This was particularly pertinent when looking at how people responded
to drugs: “There are many interesting evolutionary questions there”,
he said.
Multidrug-resistant organisms
One of the organisms that was causing great problems today was Staphylococcus
aureus in the form of methicillin-resistant S aureus (MRSA). Other organisms,
however, gave an even more lurid picture of antibiotic resistance. Multidrug-resistant
Mycobacterium tuberculosis (MDR-TB), which was resistant to the most powerful
antituberculous drugs, was one of those.
“We used to think [MDR-TB] was yesterday’s problem in this country,
but it never went away”, Professor Pennington said. It had been known for
many years that tuberculosis needed to be treated with multiple drugs but, in
practice, therapy was often inappropriate, incomplete or taken erratically.
This resulted in antibiotic resistance due to selection of naturally occurring
drug-resistant mutants of M tuberculosis. MDR-TB was very common in Russia,
and it was from there that it would, almost certainly, spread to the UK in the
not too distant future, Professor Pennington warned. MDR-TB was particularly
common in Russian prisons, which were notoriously overcrowded.
“There is a major, major problem of MDR-TB there just festering away,”
he said.
One of the areas of antibiotic resistance where little was known, was the amount
of antibiotics used over a particular period and whether or not resistance developed
by an evolutionary process in response by selection. According to Professor
Pennington, a lot of data had been collected in this area, but all rather empirically.
A large proportion of the antibiotics being used were not in human medicine.
For example, in the UK, in 1997, 323.2 tonnes of tetracycline and 119.6 tonnes
of beta-lactam antibiotics were used in food animal production.
The way forward
Discussing possible solutions to the problem, Professor Pennington remarked
that, in terms of the portfolio of antibiotics that were available at present,
researchers were getting close to the end game in terms of developing new approaches
(eg, side chain modifications).
“What we need are brand new drugs that work on sites we have not been working
on at the moment,” he said.
Also, it was important to remember that antibiotics were not the be all and
end all of treating diseases. Active national surveillance programmes were needed
to monitor antibiotic usage. Prudent antimicrobial usage was necessary to reduce
the pressure for resistance.
Have bugs, will travel
Throughout his presentation, Professor PETER HAWKEY (department of microbiology,
University of Leeds) illustrated the extreme mobility of antimicrobial resistance.
“It is the mobility in the world that is changing dramatically the whole
face of antibiotic resistance,” he said.
It was not only pathogens that were moving, but commensals as well. Another
important factor was the movement of food and people around the world.
Professor Hawkey described several new enzymes that had emerged, eg, TEM-3 and
the SHV group of enzymes, that were able to destroy antibiotics, including third
generation cephalosporins. SHV enzymes were closely related to TEM enzymes (sequence
homology about 60 per cent); both types of enzymes were subject to point mutations.
With SHV, a mutation in a single base pair could change one amino acid which,
in turn, could change the active site and enable the enzyme to destroy an antibiotic.
“This is pretty impressive stuff in terms of molecular evolution,”
Professor Hawkey said.
He described a situation which had arisen at St James’s university hospital
in Leeds where 16 strains of Escherichia coli with high-level resistance to
extended-spectrum cephalosporins and other classes of antibiotics had been isolated.
These isolates had been found to produce up to three separate beta-lactamase
enzymes, including TEM-1 and SHV-5. With the exception of the carbapenems, the
isolates were resistant to all beta-lactam antibiotics, including extended-spectrum
cephalosporins and aztreonam. Furthermore, there was evidence of the spread
of a plasmid encoding SHV-5, particularly among patients on the hospital’s
liver transplant unit. Four of the five patients carrying isolates that carried
a plasmid-located bla Amp C gene (these isolates expressed an Amp C-type beta-lactamase
which was not affected by the beta-lactamase inhibitor clavulanic acid) had
recently visited the Indian subcontinent, and it was assumed that these patients
had returned carrying these bacteria.
“This study demonstrates the ease with which highly resistant bacteria
can be imported into the UK and spread within hospitals,” Professor Hawkey
warned.
Genomics and transcriptonomics
Following on, Dr MARTIN BURNHAM (Smithkline Beecham Pharmaceuticals, US) discussed
the application of genomics and transcriptonomics to the search for new antibacterial
targets. There had clearly been a slow-down in the discovery of new antibiotic
classes over the past few decades, and certainly a slow-down in the discovery
of successful new classes, he said. There was now an urgent need for new classes
of antibacterial agents to which there were no resistance mechanisms.
The availability of multiple bacterial genome sequences representing all possible
antibacterial targets had revolutionised antimicrobial drug discovery in the
pharmaceutical industry. Smithkline Beecham’s microbial genome database
contained the sequences of over 60 bacteria, including different isolates of
the same pathogens.
He outlined how his group had identified novel antibacterial targets by comparing
whole bacterial genome sequences and selecting genes according to:
In order to conduct this prioritisation process, his group had developed technologies which allowed monitoring of gene transcription during infection and control of gene expression either in vitro or in infection to assess their relevance to viability or pathogenesis. According to Dr Burnham, following the adoption of a gene product as an antibacterial target, and high-throughput screening to identify inhibitors, these same technologies were invaluable in confirming the mode of action of antibiotic leads and in guiding their subsequent chemical development to achieve a useful potency and spectrum of activity.
Targeting resistance mechanisms
Professor KEITH POOLE (department of microbiology and immunology, Queen’s
university, Kingston, Canada) described approaches to targeting resistance mechanisms.
While resistance mechanisms almost certainly predated clinical usage of antibiotic
agents, the extensive use of these agents has tended to select and, thus, enrich
for bacteria expressing resistance determinants. Current emphasis was on identifying
new targets and developing new antibacterial agents, in which case there would
be no need to worry about current problems of resistance.
The problem with using only this approach was that newer problems of resistance
would continue to evolve and develop. Rather than constantly trying to develop
new agents, a second approach was to tackle the problem head on by developing
mechanisms of short-circuiting or circumventing resistance mechanisms themselves
and, essentially, rejuvenating currently available antimicrobial agents.
Professor Poole classified resistance mechanisms into:
Antibiotic modifying enzymes
One of the best targets with regard to inhibiting resistance mechanisms as a
way of enhancing the utility of currently available antibiotics were antibiotic
modifying enzymes, Professor Poole said. The best example of these was the aminoglycoside-
modifying group of enzymes, which were the major determinant for aminoglycoside
resistance. Three classes of aminoglycoside-modifying enzymes existed: phosphoryltransferases
(APH), acetyl transferases (AAC) and adenyltransferases (ANT).
Two approaches had been taken to try to counteract APH-mediated resistance,
which was the most important determinant of aminoglycoside resistance. One approach
depended on the use of inhibitors that blocked APH enzyme activity; the other
approach, which Professor Poole referred to as “APH immunity”, was
to develop aminoglycoside-like molecules that were not affected by APH. In terms
of APH inhibitors, two types had been described: substrate-derived (aminoglycoside-like
molecules that were recognised by the enzyme and act at the active site), and
non-substrate-derived APH inhibitors. In vitro studies using both these types
of APH enzyme inhibitors had shown promise, although they had not yet rendered
APH-expressing organisms susceptible to aminoglycosides.
Targeting efflux mechanisms
Some of the other approaches that could be used to target resistance mechanisms
included efflux systems.
With regard to agent-specific (eg, tetracycline) efflux systems, one approach
was to avoid efflux by developing agents that were not good substrates for the
efflux system, eg, glycylcyclines, or to develop efflux inhibitors.
Efflux systems which could accommodate multiple agents (eg, Nor A of S aureus)
are particularly effective determinants of fluoroquinolone resistance. Inhibitors
of Nor A had been identified, eg, 5¢-methoxyhydnocarpin (5¢-MHC) from
Berberis spp. Such inhibitors were capable of enhancing the susceptibility of
efflux-positive strains, and also of compromising the emergence of resistance
in susceptible strains.
Summing up, Professor Poole suggested that since antibiotic resistance was probably
unavoidable, the activities of existing and, as yet, undiscovered antimicrobials
through resistance inhibition might be crucial for continuation of the antibiotic
era.
Antibiotic resistance: the way forward
The second part of the symposium, chaired by Professor Stephen Denyer (University
of Brighton) and Dr Geoff Hanlon (University of Brighton), focused on solutions
to the global threat of antimicrobial resistance.
The first speaker, Professor PAUL WILLIAMS (institute of infections and immunity,
University of Nottingham) discussed targeting virulence as a means of attenuating
infection.
Professor Williams explained that pathogenic bacteria possessed distinct genetic
properties which confered a significantly greater capacity to compete with other
(commensal) bacteria to gain a foothold within a susceptible host, to multiply
in host tissues and to avoid host defences. Pathogens had therefore evolved
complex regulatory pathways to control virulence determinant production. The
ability to switch off virulence and survival gene expression by targeting these
regulatory pathways offered an alternative strategy for the treatment and prevention
of bacterial infection. Rather than killing the infecting micro-organism, such
antibacterial agents would attenuate virulence such that the pathogen failed
to adapt to the host environment, and could readily be cleared by the host defences.
However, until recently, this approach had lacked specific targets for rational
drug design.
Quorum sensing
According to Professor Williams, the discovery that bacterial cells communicated
with each other using diffusible signalling molecules to determine (in concert
with bacterial cell population density) when to deploy their virulence determinants
offered such a target. This regulatory mechanism was known as “quorum sensing”.
The challenge was to find ways of interfering with the synthesis of the quorum-sensing
signal molecule, or to antagonise the interaction between the signal molecule
and its receptor. Both of these approaches would have the effect of switching
off virulence gene expression and thus attenuating the pathogen.
In order to develop quorum-sensing blocking agents (QSBs), it was necessary
to understand the molecular mechanisms by which quorum-sensing signal molecules
were generated. For example, Pseudomonas aeruginosa and Staphylococcus aureus
used N-acylhomoserine lactones and modified cyclic peptide thiolactones, respectively,
as quorum-sensing signal molecules.
Concluding, Professor Williams said that quorum sensing offered a novel target
for anti-infective therapy with the potential to circumvent the current problems
posed by resistance to conventional antibiotics. However, there were also several
likely disadvantages of QSBs, such as a narrow spectrum of activity, non-bactericidal
effect, questionable efficacy in immunocompromised patients, and the need for
development of new diagnostic systems since MIC (minimum inhibitory concentration)
testing would not be useful with this approach.
Studying bacteria in vivo
Dr CHRISTOPH TANG (Medical Research Council clinical scientist, John Radcliffe
hospital, Oxford) described techniques that had been developed for studying
bacterial genes essential for growth in mammalian hosts.
According to Dr Tang, such techniques either examined the profile of gene expression
at different stages of disease (eg, in vivo expression technology, IVET), or
used a mutational approach to define genes required for survival of bacteria
in the host (signature-tagged mutagenesis, STM). IVET was a strategy for identifying
promoters whose expression was induced in specific environments during pathogenesis.
STM involved the labelling of mutants with unique DNA sequence tags that allowed
the identification of individual mutants within a pool. This allowed high-throughput
screening of libraries of insertional mutants in animal models of infection.
According to Dr Tang, a wide range of microbes had been examined using these
techniques in diverse host environments. This work had provided a broad view
of the process of pathogenesis, and had emphasised the importance of nutrient
acquisition and cell wall integrity during infection. The question was how to
go from gene identification to identifying new targets and new molecules which
would have a global impact on infectious disease.
New drugs from old bugs
The final speaker of the symposium, Professor JIM STAUNTON (department of chemistry,
University of Cambridge) discussed a biotechnology approach to building new
antibiotics.
His group’s approach was to use combinatorial biosynthesis to develop “new
drugs from old bugs” by speeding up the evolution of natural product diversity.
Its goal was to produce hybrid structures from hybrid organisms. About 20 macrolide
structures had been produced from this forced evolution, but only a small proportion
had been further developed. According to Professor Staunton, the activity of
one compound had equalled that of a marketed antibiotic. Looking to the future,
Professor Staunton said: “Somewhere among all this activity, some very
useful compounds are going to come out.” — Contributed.