By David N. Leff

Four years ago, at the University of Alabama, in Birmingham, infectious-disease clinicians suspected multi-drug resistance by the microbe Klebsiella pneumoniae to various antibiotics they were using for treatment of urinary tract infections.

They sent these suspect strains for analysis to microbial geneticist George Jacoby, head of infectious diseases at the Lahey-Hitchcock Clinic, in Burlington, Mass.

"At the time," Jacoby told BioWorld Today, "they did not realize they had an unexpected problem with quinolone antibiotics. Those bugs from Alabama turned out to have an unexpected kind of resistance, which may in the future compromise treatment of infections with quinolones." (These quinolone antibiotics include ciprofloxacin, norfloxacin, ofloxacin and nalidixic acid.)

"Receiving those Alabama isolates was a fortuitous discovery," he said.

At the time, Jacoby was doing a collaborative study with a colleague, Luis Martinez-Martinez, a microbiologist at the University of Seville, in Spain.

Their project involved putting microbial plasmids that carry a particular type of resistance to beta-lactam antibiotics — such as penicillin — into strains of Klebsiella.

"Everybody knew," Jacoby recounted, "that plasmids don't determine quinolone resistance. In 1987, a report in The Lancet said in effect, 'Aha! We have found a quinolone resistance plasmid.' But the evidence didn't support it.

"My colleague in Seville," Jacoby went on, "was looking at quinolone resistance as a control for the beta-lactam resistance he was supposed to be studying. And he found, by golly, that in one of the 20 strains that he examined, the bug appeared to be quinolone-resistant. I didn't believe it, because it wasn't supposed to happen.

"So he sent the strain back to me. I checked it out, and we looked at the data some more — and I did believe it."

Resistance Gene Lurked In Multiresistance Plasmid

The current issue of The Lancet, dated March 14, 1998, carries an "Early Report" by Jacoby and Martinez-Martinez, titled: "Quinolone resistance from a transferable plasmid."

Bacteria, besides their main chromosomal genomes, carry small loops of DNA called plasmids. The one that apparently caused the quinolone drug resistance, Jacoby said, "is a big hunk of circular DNA, 56 kilobases in size. It would have room for 50 or 60 genes."

Jacoby, whose research laboratory is at the Veterans Administration Medical Center, in Bedford, Mass., plucked the 900-base gene for quinolone resistance out of that plasmid and cloned it. He's now working on sequencing it.

"We have an idea," Jacoby said, "that it isn't big enough to be a gyrase gene. Gyrase is the bacterial enzyme that is targeted by quinolone action."

His paper describes how that plasmid amplified the microbe's resistance to ciprofloxacin, a quinolone-derived antibiotic, and passed it on to other infectious bacteria by conjugation.

"In the presence of the plasmid," he recounted, "we observed an initial eight-fold increase in minimum inhibitory concentration (MIC) of ciprofloxacin — up to 32 micrograms per milliliter. MIC is the concentration of antibiotic at which the bacteria won't grow. But when we moved that plasmid into a strain of Escherichia coli, the MIC went down to 0.25. That's still an increase, but it's not even a level that could be picked up in a clinical lab as resistant."

Jacoby determined that, although low in itself, the quinolone resistance enhanced the ability of the bacterium's chromosomal DNA to express resistance mutations, such as limiting the ability of the antibiotic to get into the cell, or changing its target there.

"We looked to see," he went on, "what interactions this plasmid-mediated resistance mechanism would have with the chromosomal resistances that we knew occurred. And we found that they didn't cancel each other out. The resistances worked together with at least an additive effect."

Normally, bacteria have tiny pores, called porins, all over their outer membrane. These are ports of entry for antibiotics, but can be abolished by chromosomal mutation. Other mutations alter the DNA gyrase and topoisomerase enzymes, which quinolone targets.

"If the plasmid is now present," Jacoby observed, "the resistance frequency jumps up at least 100-fold, and you can sequentially select even higher levels of resistance that could be clinically important."

Quinolones Still Effective Drugs

He pointed out that "one of the reasons quinolone antibiotics are very widely used is that they have been effective against some of our problem pathogens, which have become resistant to other antibiotics. Quinolones are available orally as well as intravenously, and work against all sorts of Gram-negative and, recently, Gram-positive organisms."

Microbial resistance to the quinolones has not yet taken off in the U.S., Jacoby observed. "But it does put up a red flag that bacteria have another trick up their sleeve, which is going to facilitate their ability to get around an otherwise excellent class of antibiotics."

That red flag was run up the flagpole last week in Atlanta at an international meeting on new and emerging diseases. Jacoby had attended a preview session the weekend before.

Participants heard from the Centers for Disease Control, co-sponsors of the gathering, that 2 million Americans a year catch infections in hospitals, of which 70 percent are due to microbial antibiotic resistance.

As for prospective countermeasures, Jacoby pointed out, "We know well that resistance mechanisms involve enzymes that inactivate, or fit with, the antibiotics. They have to make contact with the drug, at an active site, with the right configuration, charge and binding properties, so they can work on the substrate.

"If you have a situation like that," he suggested, "you can potentially modify your substrate, put an extra side-chain on your antibiotic, or find something that will prevent the enzyme from attaching, without preventing the drug from doing its job. It's also possible to find another chemical that blocks the resistance mechanism."

He continued: "When we know the quinolone resistance chemistry a little better, we may be able to modify the antibiotic rationally, or potentially find an inhibitor. At this point," he concluded, "we're talking about doing the right experiments — but they haven't been done yet." *

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