New weapons in the fight against corneal infection

By Laszlo Dosa

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.

A major 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 corneal infection.

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.

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