New weapons in the fight against corneal infection
WASHINGTON, DC - Potent new antibiotic agents which inhibit cell
wall synthesis and bacterial vaccines are among the most promising
developments in the pipeline for the prevention and treatment of
ocular infection, according to Richard J. O'Callaghan PhD.
Dr O'Callaghan has focused his work on Staphylococcus aureus, particularly
the drug resistant strains. He discussed his ongoing research at
a seminar sponsored by the Research to Prevent Blindness Foundation.
His in vivo studies suggest Moxifloxacin, a new fluoroquinolone
antibiotic, may have a role in the treatment of corneal infections.
He injected the bacteria into the cornea of rabbits and then waited
four hours before applying drops of different antibiotics every
hour until nine hours post-infection. Next, he counted the bacteria
still alive in the cornea one hour after the last drop.
"We find that the untreated eye has just over one million bacteria.
If we treat it with Vancomycin, we see the number of bacteria reduced
to hundreds per eye. If this is one of those tough strains and we
treat it with Levofloxacin, we bring it down.
"But if we treat it with Moxifloxacin, it reduces it almost
to zero. And Moxifloxacin will work on many strains while Levofloxacin
will not," he explained.
Dr O'Callaghan's studies indicate that Moxifloxacin is a very powerful
antibiotic that will work on a drug-sensitive strain and on a high
percentage of the most resistant strains.
The drug may become available in the next year or so. But sooner
or later Staphylococcus will become resistant to any antibiotic
so the search is on for a different approach, he said.
Dr O'Callaghan believes he may have found something along these
lines in a protein compound called enzybiotic.
Antibiotics work by blocking a pathway in the bacterium. Cipro,
for example, interferes with DNA synthesis of the bacterium and
Gentamicin blocks protein synthesis.
But enzybiotics, on the other hand, do not block any synthetic reaction.
Instead, they digest the bacterial cell wall.
Bacteria have very concentrated amounts of protein in the cytoplasm
and very little water. When the bacterium is in the blood, osmotic
pressure forces the fluid to try to rush in, keeping the cytoplasm
under water pressure at all times.
Like a balloon, it is constantly being blown up. Staphylococcus
is covered by the cell wall like a steel cage that holds it together.
When an enzybiotic digests the bacterial cell wall, the bacterium
can no longer resist the water pressure and it explodes.
Dr O'Callaghan explains that an enzybiotic is 30 times larger than
a typical antibiotic. When he added an enzybiotic called Lysostaphin
to bacteria, it chews up the cell wall until its loses its strength
and the membrane bulges and pops under the water pressure. It appears
to be a very powerful new way of killing bacteria.
"There is a lot of optimism that the organism will remain sensitive.
The downside of a molecule like Lysostaphin is that being a protein
- a big molecule - it can cause an immune response to itself.
"And we are worried there might be an allergic reaction if
you give the patient the molecule several times. But we have not
experienced that in animals," Dr O'Callaghan said.
He applied six drops of Lysostaphin to rabbit corneas and several
hours later the number of bacteria dropped to zero.
With Vancomycin, the bacteria count reduced to 100. Lysostaphin
appears to be very powerful in a much lower concentration than Vancomycin.
advantage of Lysostaphin is that it is very specific in its reactivity.
It can kill only Staphylococcus aureus. This product is not going
to work if the patient has a different kind of infection
"I regard this compound as a sort of an ace in the hole that
works. If Staphylococcus ever becomes resistant to Moxifloxacin
and there is no other antibiotic available, this is something we
can develop rapidly and use on patients with really horrible strains
of bacteria. If Moxifloxacin ever fails us before we have another
antibiotic, we could maybe use Lysostaphin," he noted.
Dr O'Callaghan's long-term goal is also to develop a vaccine against
For nearly 10 years his lab has been trying to identify the proteins
the bacteria make which damage the eye.
The challenge was to find a specific bacterial strain and trigger
a mutation in its DNA so that it cannot make a particular protein.
Then placing that bacterium into the eye and letting those mutants
grow without making that particular protein, the bacterium may lose
some of its ability to destroy the tissue. It appears that the search
has been fruitful.
"In Staphylococcus there is a molecule called alpha-toxin.
When we mutated it, we saw the tissue damage drop by about 60%.
We can take the parent strain which has the gene for alpha-toxin,
a normal gene, and receive a clinical high score.
"If we put the mutant into the eye of the rabbit, it's a much
lower score. We can take that mutant and add a functional copy of
that gene back, called a rescue bacterium, and see that it is really
a singular protein," Dr O'Callaghan said.
He purified the protein and injected 50 nanograms into the rabbit
eye. It was found to be toxic to the cornea, destroying the epithelium
and causing much tissue damage.
The protein then was treated to retain its immunogenecity but lose
its toxicity. This modified protein is called the toxoid and forms
the basis for an experimental vaccine.
When it is injected subcutaneously into the rabbit, the animals
appear to develop an antibody-based immunity. Inspection of the
eyes of rabbits revealed that in the non-immunised ones which have
been infected, tissue damage occurred within 25 hours.
In those which were immunised, tissue damage was less than half
as high. Moreover, there was very little epithelial damage in the
immunised rabbits which is important to the ophthalmologist concerned
with the healing of the cornea.