Why so persistent?

Staphy­lo­coccus cells. Photo by Think­stock photos.

Per­sis­tence — it’s what keeps us all sur­viving. If it weren’t for this lovely quality, we’d just give up and crawl under a rock some­where because it’s all just so darn dif­fi­cult out there in the world. Same’s true for every bac­te­rial infec­tion we know of, the chronic ones in par­tic­ular. Per­sis­tence is para­mount. Think about it: we humans do every­thing we can to get rid of these fero­cious microbes, but try as we may, many of them just refuse to die. It’s one of the most pressing issues in public health today. From biofilms on med­ical devices to the ever-​​dreaded staph infec­tion, sev­eral per­sis­tent bac­te­rial infec­tions elude cur­rent med­ical wisdom.

Ear­lier in his career, Kim Lewis of the Col­lege of Sci­ence showed that a select few bac­te­rial cells stick around after antibi­otic onslaught even though they are genet­i­cally iden­tical to their not-​​so-​​lucky com­pa­triots that suc­cumb. Lewis has ded­i­cated his life to fig­uring out what makes these per­sister cells, as they’re called, so good at sur­viving. Since most antibi­otics work by dis­rupting active processes of cel­lular func­tion, it seems as though per­sis­tence is akin to dor­mancy. For instance, if a cell isn’t making DNA because it’s “asleep,” then a drug that works by inter­fering with DNA pro­duc­tion and causing damage won’t have any impact on that par­tic­ular cell. What sends a cell into dor­mancy remains a mys­tery, though. About a decade ago Lewis’ team showed that pairs of bac­te­rial pro­teins called toxin/​antitoxin pairs seem to mediate dor­mancy by stop­ping and starting cell growth at var­ious points.

A few years later, the team iden­ti­fied a spe­cific toxin/​antitoxin pair  that can be induced when a cer­tain class of antibi­otics, called flu­o­ro­quinolones, comes on the scene. “It’s very clever,” said Iris Keren, a senior research sci­en­tist in the Lewis lab. “If you treat a patient with flu­o­rquinolones, yes you kill a lot of bac­teria, but at the same time you actu­ally help them gen­erate more per­sis­tors.” Flu­o­ro­quinolones act by dam­aging bac­te­rial DNA, a pretty stressful sit­u­a­tion for the bugs. Another form of stress that’s par­tic­u­larly dif­fi­cult for bac­teria is called oxida­tive stress. When bad bac­teria get into the body, the immune system responds by sending a macrophage to eat them up in big gulps. When the macrophages see their dinner, they start sending out so-​​called “reac­tive oxygen species” and “reac­tive nitrogen species.” The bac­teria respond with their own global defense system, elim­i­nating the reac­tive species and trying to repair damage. It seemed rea­son­able to think that such a response may also cause some of the cells to go into dor­mancy and become per­sis­ters. So, they hit the bench.

First, they exposed bac­te­rial cul­tures to a com­pound called paraquat, which causes oxida­tive stress sim­ilar to macrophages. Then they tried to kill the bac­teria with three dif­ferent antibi­otics, one of which was a flu­o­ro­quinolone (oflaxacin to be spe­cific). The other two were ampi­cillin, which is a type of beta-​​lactam and works by pre­venting proper cell wall syn­thesis, and kanamycin, an amino­gly­co­side which affect pro­tein syn­thesis causing the gen­er­a­tion of aber­rant toxic proteins.

The researchers were sur­prised by their results: The number of per­sister cells in the cul­ture did spike after oxida­tive stress — but only in the case of one of the three drugs: the flu­o­ro­quinolone. Oxida­tive stress had no affect on per­sister levels in the case of the other two antibi­otics. Clearly, some­thing else was going on.. The team decided to take a closer look at the per­sister cells that came out of the flu­o­rquinolone expo­sure exper­i­ment. It turned out these cells were now not just tol­erant to that drug, they were also tol­erant to both ampi­cillin and kanamycin. So cells that were treated with paraquat and than with ofloxacin were now tol­erant to all three antibiotics.

Before we keep going, we first need to under­stand why the flu­o­rquinolone behaved dif­fer­ently that the other antibi­otics. Paraquot (the com­pound used to cause oxida­tive stress) also induces some­thing called a mul­tidrug resis­tant (MDR) pump. It’s a type of pro­tein that resides in the cell wall and is capable of pumping out dif­ferent kinds of drugs that make their way into the cell. While it does work on dif­ferent types of drugs, it has no affect on beta-​​lactams or amino­glyo­sides. Of the three antibi­otic types that the team threat­ened their little bac­te­rial cul­tures with, only the flu­o­rquinolones were actively removed by the MDR pump. This is classic antibi­otic resis­tance. The bac­teria is pre­sented with some­thing that’s trying to kill it and it actively sweeps that toxin out of the house.

Per­sis­tence is much dif­ferent. Those cells that happen to be in a dor­mant state for one reason or another tol­erate all antibi­otics they’re attacked with. When they wake back up, if the antibi­otic is still around the per­sis­ters will indeed die. But if the antibi­otic is gone, sweet! They can rekindle their life­long dream of reproduction.

So, first the oxida­tive stress turned on the MDR pump, which made it resis­tant to the flu­o­ro­quinolone. Then, expo­sure to that drug sent most of the cells into a dor­mant state which pro­tected them  from the other antibi­otic, explained Keren. “This is an inter­esting finding,” said Keren, because we now get into this inter­sec­tion between per­sis­ters and resistance.”

Under normal con­di­tions, per­sis­ters are still a problem, but this series of events increased the number of per­sis­ters around. While the work is invalu­able in helping researchers to under­stand a little more about how per­sis­tence works, it could also prove a useful tool for fur­ther study. One of the biggest issues in studying per­sister cells is their rel­a­tive obscu­rity. In a colony of bac­teria, only a small frac­tion are usu­ally per­sis­ters so iso­lating them and studying them is dif­fi­cult. But with pro­to­cols such as this to induce per­sis­tence, researchers have more to work with.