For the first time, researchers have discovered strains of a deadly, multidrug-resistant bacterium that uses a cryptic method to also evade colistin, an antibiotic used as a last-resort treatment. That’s according to a study of US patients published this week by Emory University researchers in the open-access microbiology journal mBio.

The wily and dangerous bacteria involved are carbapenem-resistant Klebsiella pneumoniae or CRKP, which are already known to resist almost all antibiotics available, including other last-line antibiotics called carbapenems. The germs tend to lurk in clinical settings and can invade the urinary tract, bloodstream, and soft tissues. They’re members of a notorious family of multidrug-resistant pathogens, called carbapenem-resistant Enterobacteriaceae (CRE), which collectively have mortality rates as high as 50 percent and have spread rapidly around the globe in recent years. A 2013 report by the Centers for Disease Control and Prevention estimated that there were more than 9,300 CRE infections in the US each year, leading to 600 deaths. Both the CDC and the World Health Organization have listed CRE as one of the critical drug-resistant threats to public health, in need of "urgent and aggressive action."

That’s what we knew about CRKP before this week.

In the new study, the Emory researchers discovered two strains of CRKP—isolated from the urine of patients in Atlanta, Georgia—that can also resist colistin. But they do so in a poorly understood, surreptitious way. At first, they appear vulnerable to the potent antibiotic in standard clinical tests, but with more advanced testing and exposure to the drug, they reveal that they can indeed survive it. In mice, the strains caused infections that couldn’t be cured by colistin and the mice died of the infections. Mice infected with typical CRKP were all saved with colistin.

So far, there’s no evidence of CRKP infections surprisingly turning up resistant to colistin during treatment in patients. But the authors, led by microbiologist David Weiss, say that may be because the evidence is difficult to gather, and the data so far is cause for concern. The researchers concluded that the findings "serve to sound the alarm about a worrisome and under-appreciated phenomenon in CRKP infections and highlight the need for more sensitive and accurate diagnostics.”

Deadly riddle

In an interview with Ars, Weiss emphasized the CRKP’s stealth resistance is “obviously most concerning when it’s to a last-line drug,” such as colistin. “The patients who could be treated don’t have many options at this point.”

And the dire situation is what makes the problem hard to study in the clinic. For instance, by the time most patients with CRKP infections get to the point of needing colistin, they’re usually extremely sick, Weiss adds. In those cases, doctors tend to throw as many antibiotics at patients as they can, which makes it tricky to tell if stealth colistin resistance is a problem. “I’m not saying if it were me I wouldn’t want a whole bunch of antibiotic drugs,” he said. But to really know if this hidden colistin resistance is a problem, you’d have to be using a single therapy of just colistin.

In the lab, however, the researchers can explore the cryptic resistance, called hetero-resistance. Weiss, director of the Emory Antibiotic Resistance Center, has been studying hetero-resistance for several years, but the phenomenon is still a bit of a mystery.

When microbiologists get bacterial isolates from patients, they grow them up in big batches of nutrient broth. This generally results in a genetically identical population of bacteria that—usually—have uniform set-points of resistance or susceptibility to a certain antibiotic. In other words, if they’re susceptible, a relatively low concentration of the drug will kill off the population. If the bacteria can withstand higher doses, they collectively enter resistance territory. Resistance can occur along a spectrum, but there are often standardized thresholds for determining that a bacterial strain is resistant. In other words, if the population on the whole survives “X” concentration of a certain antibiotic, it’s then considered resistant.

Hetero-resistant populations don’t play by these rules. In standard diagnostic tests, the population may look completely susceptible. But in advanced tests, researchers can detect sub-populations that are resistant. Usually, this might suggest that there were just some contaminating bacteria that had a genetic element that protected them from the drug. But these sub-populations in hetero-resistant bacteria appear genetically identical to their susceptible counterparts. They’re clones of each other that for some reason are doing something different to be resistant to the drug.

Underhanded microbes

This was the case for the CRKP clinical isolates that Weiss and his colleagues collected and studied. Standard tests for resistance suggested that colistin concentrations of 0.5 μg/mL or less could kill the populations—they were susceptible. But further experiments found that 1 in 1,000 cells could survive 2μg/mL of colistin. And 1 in 1,000,000 survived 100μg/mL.

When the researchers grew up the population in colistin, the resistant subpopulations took over. But switching the populations to broth without colistin, the population reverted to susceptible again—except for the 1 in every 1,000 cells. The researchers also sequenced the genomes of bacteria resisting colistin and those susceptible to it. They were genetically identical.

Even though they have the same blueprints, they’re activating their genes differently, Weiss says. It’s unclear how or why this happens. So far, Weiss and his colleagues hypothesize that a specific sensory system may be key to the resistance trick. The system—a type of two-component signal transduction system—involves a protein embedded in the bacteria’s membrane that responds to some environmental cue. That protein can then pass the signal to another protein inside the cell that then switches genes on or off accordingly.

This system appeared turned “on” in some of the resistant sub-populations. And when researchers used genetic engineering to break the system in other hetero-resistant bacteria, the populations lost their mysterious antibiotic evasion.

It’s unclear what genes the system is controlling, but evidence so far suggests that genetic tinkering allows the cell to make its outer membrane less negatively charged to thwart colistin. The antibiotic is positively charged and seems to kill by breaking down bacterial cells' otherwise negatively charged outer membranes. But the precise mechanism behind this and the bacteria’s defenses are still unclear.

“I think we got to a place [with antibiotics] where [we said] ‘oh that one stopped working? Who cares? We’ll use the next one,’” Weiss said. “So, there wasn’t this urgency to understand all of the details of it because we had back-up options. Now that we’re running out of back-up options, people are much more interested.”

Overall, “there’s a bunch we don’t know still,” he said, emphasizing the need for more research funding. “But we’re working on it.”

mBio, 2018. DOI: 10.1128/mBio.02448-17 (About DOIs).