Evolution of antibiotic resistant bacteria

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Evolution of antibiotic resistant bacteria
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Tremendous quantities of antibiotics are produced
and released into the environment. 90 180
million kg/year of antibiotics are used (enough
for 25 BILLION full treatment courses 4 per
human/yr!)About 10 X more antibiotics are used
in agriculture than to treat people. (Levy 1997
estimated 30 X more in animals than in people).
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• There are two major effects of an antibiotic
  therapeutically, it treats the invading
  infectious organism, but it also eliminates
  other, or non-disease producing, bacteria in its
  wake. The latter do, in fact, contribute to the
  diversity of the ecosystem and the natural
  balance between susceptible and resistant
  strains.

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• The consequence of antibiotic use is, therefore,
  the disruption of the natural microbial ecology.
  This alteration may be revealed in the emergence
  of types of bacteria which are very different
  from those previously found there, or drug
  resistant variants of the same ones that were
  already present.
• Levy, 1997

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• Alexander Fleming (discoverer of penicillin)
  recognized the potential danger of antibiotic
  resistance.
  
• In 1945, he warned that misuse of penicillin
  could lead to the selection and propagation of
  mutant forms of bacteria resistant to the drug.
  
• The first penicillin-resistant bacteria appeared
  several years later. Their mutant gene encoded
  for a penicillin-destroying enzyme, penicillinase.

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"... the mounting use of antibiotics, not only in
people, but also in animals and in agriculture,
has delivered a selection unprecedented in the
history of evolution." Levy, 1997
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The development of penicillin resistance in
gonococcal populations from 1980 through 1990 was
incredibly rapid.
Figure 6.8 in Atlas and Bartha, 1998
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Other examples In Japan, in 1953, only 0.2 of
Shigella (causes bacillary dysentery) was
resistant to antibiotics. By 1965, 58 were
resistant to sulfnilamide, streptomycin,
chloramphenicol, and tetracycline.
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Penicillin was extensively used in Hungary in the
early 1970's. By 1976, 50 of the strains of
Streptococcus pneumoniae were resistant to
penicillin.
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Intensive care units seem to have particularly
high incidences of resistant microbes.
Figure 10.27 from Atlas, 1997
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Bacteria become resistant through

• Adaptations (product of selection)
• Acquisition and transmission of antibiotic
  resistance (horizontal gene transfer)

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Evolution through natural selection can occur
remarkably quickly when selection pressures are
very strong and reproductive rates are very fast
(some bacteria generations are as short as 15-20
minutes!)
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• Genes that code for antibiotic resistance were in
  the gene pool before humans began to produce
  antibiotics over 50 years ago.

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This was shown clearly by some experiments by
Joshua Lederberg.
Fig 7.3 Volpe and Rosenbaum, 2000
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• Mutations furnish the source of genetic
  variability and natural selection acts upon that
  variability to generate adaptation (antibiotic
  resistance)

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Mechanisms of resistance

• Decreased transport of the antibiotic into the
  cell membrane.
  
• Production of enzymes that destroy the inhibitory
  capacity of the antibiotic (e.g. by hydrolyzing
  it so that it loses its inhibitory ability).

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Mechanisms of resistance

• Modification of the antibiotic binding site so
  that the drug no longer binds to the target.
  
• Production of alternate molecules that can
  replace those disrupted by the antibiotic.

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Mechanisms of resistance

• Production of mechanisms to pump antibiotics out
  of cells
  
• Production protective biofilms (upper layers
  protect lower layers)

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Acquisition and transmission of antibiotic
resistance

• Bacteria often exchange resistance genes through
  R plasmids

Fig 7.4 Volpe and Rosengaum, 2000
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• Up to a thousand plasmid copies may exist in a
  cell and each one may carry as many as 300
  different genes.

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Acquisition and transmission of antibiotic
resistance

• Transposons can move small DNA elements
  (including resistance genes) into bacterial
  chromosomes and/or bacteriophages.

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Acquisition and transmission of antibiotic
resistance

• R plasmids spread easily from one bacterium to
  even across species because they are conjugative
  plasmids, meaning that they not only code for
  antibiotic resistance but also for mating, which
  increases the rate of transfer.

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• Antibiotics are probably driving the evolution
  and spread of resistance plasmids

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Fig 10.28 Atlas, 1997
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• Stuart Levy has conducted many studies of
  antibiotic resistance in E. coli associated with
  chickens.
  
• When chickens that hosted E. coli with
  multi-resistance plasmids were kept in a clean
  and isolated part of the barn, they did not lose
  the resistant bacterial strains over many months
  of the study.
•  

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• But, when the chickens cages were relocated to
  different sites around the barn, then the
  incidence of antibiotic resistant E. coli was
  slowly reduced in the chickens microflora
  community.
• In another study, 4 chickens excreting resistant
  flora were added to 10 chickens excreting
  susceptible flora. Resistance was lost, the
  susceptible flora won out.
•  

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• What was the role of adding chickens that
  harbored susceptible microflora?
  
•  

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• For immediate change in resistance frequency,
  the result relies on numbers, not large
  differences in bacterial fitness. Moreover,
  there is no active counter-selective force which
  propels repopulation with susceptible strains.
• Levy 1997
•  

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• Coevolution An intimate and interactive
  evolutionary relationship between two or more
  species in which direct genetic change in one
  species is attributable to genetic change in the
  other(s).

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The Red Queen Hypothesis

• In 1973 VanValen referred to the difficulty faced
  by species locked in a coevolutionary arms race
  as the Red Queen Problem. Recall in Lewis
  Carrolls Red Queen who had to run faster and
  faster just to stay in place.
• This is an appropriate analogy the environment
  constantly changes and populations must continue
  to evolve to survive.

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Antibiotics are now everywhere in the
environment, and humans and bacteria are engaged
in an arms race.Who is likely to win?
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• perhaps the very way we fight infection should
  be reconsidered. As in other aspects of our
  social behavior, we identify sometimes-annoying
  creatures as mortal enemies and are determined to
  annihilate them.
• Amabile-Cuevas, 2004

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Therapies in the post-antibiotic era

• May target virulence factors instead of the
  entire organism.
  
• Develop vaccines to prevent infection in the
  first place.
  
• Analogous to biological control and integrated
  pest management strategies used in agriculture
  and manipulate competitors or parasites of
  virulent organisms?

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Therapies in the post-antibiotic era

• May target virulence factors instead of the
  entire organism.
  
• e.g. Design drugs that target the adhesion of
  virulent bacteria to a tissue. These drugs would
  have the advantage of slowing selection for
  resistance because they would not kill the
  bacteria.

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Therapies in the post-antibiotic era

• May target virulence factors instead of the
  entire organism.
  
• e.g. Develop drugs that target the plasmids that
  contain the resistance genes. This would be
  appropriate in the treatment of Bacillus
  anthracis in which the virulence factor is
  contained on a plasmid.

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Therapies in the post-antibiotic era

• Develop vaccines to prevent infection in the
  first place.
  
• DNA and protein sequences can reveal potential
  drug targets and facilitate the production of
  vaccines.
  
• e.g. The genome sequence of Neisseria
  meningitiidis is helping to identify candidates
  for a vaccine against this organism

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Therapies in the post-antibiotic era

• Manipulate competitors or parasites of virulent
  organisms.
  
• e.g. establish healthy communities of
  microorganisms in ears and gastrointestinal
  tracks

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• ... Some have talked about spraying hospital
  rooms with susceptible commensal organisms to
  replace and compete with the disease agents."
  
• Levy, 1997

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Phage Therapeutics International Inc. is a public
Washington company formed to develop,
manufacture, and acheive regulatory approval of
phage pharmaceutical products for the treatment
of antibiotic-resistant and other bacterial
infections. They are currently performing studies
to establish the safety and efficacy of phage
treatments against Staphylococcus aureus and S.
epidermis.
41
Phage have several advantages over traditional
antibiotics. One advantage is that phage multiply
exponentially, just like bacteria. A small
initial dose of phage will multiply as it infects
cells, diminishing the need for repeated
administrations. Phage can also mutate during
replication, just as bacteria do
42
Thus, the same mechanism that may lead to
antibiotic or phage resistant bacteria can
produce new phage that recognize altered
bacteria. One side-effect of traditional
antibiotics is the killing of useful bacteria,
such as those that help us digest our food or
compete with more dangerous bacteria. The
specificity of phage reduces the chance that
useful bacteria are killed when fighting an
infection.
43
Studies in mice show protection against
otherwise lethal infections. Following an
independent test of safety and efficacy under
cGLP (current Good Laboratory Practices)
guidelines, a financing round will precede Phase
I clinical trials. Patent claims include phage
compositions, production methods, and uses for
treating diseases in humans and animals. Patents
are also being prepared for novel producer
bacterial strains for mass production of
therapeutic phage product candidates.