Researchers at Emory Antibiotic Resistance Center have shown how combinations of antibiotic drugs can destroy multidrug-resistant bacteria that demonstrate heteroresistance, a poorly understood form of bacterial antibiotic resistance that is hard to detect using conventional tests. The collective results from in vitro tests using patient-derived multidrug-resistance superbugs, and studies in live mice, suggest that combinations of existing drugs may still be effective against heteroresistant bacteria that can survive treatment using the same antibiotics given singly.

“We’re saying: don’t toss those drugs in the trash, they may still have some utility,” commented David Weiss, PhD, director of the Emory Antibiotic Resistance Center and associate professor of medicine (infectious diseases). “They just have to be used in combination with others to do so.” Weiss and colleagues report their findings in Nature Microbiology, in a paper titled “Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control infection.”

Antibiotic-resistant bacteria are a major global health threat, with one estimate suggesting that by 2050 they will cause 10 million deaths around the world every year, which is higher than the death toll due to cancer, the authors wrote. Among antibiotic-resistant bacteria, carbapenem-resistant Enterobacteriaceae (CRE), including species of Enterobacter, Klebsiella, and Escherichia, have over the past 20 years become an “urgent public health threat,” with invasive infections causing a mortality rate of up to 30%. “Some CRE isolates are resistant to all available antibiotics and there is a lack of therapeutic options to treat such infections,” the researchers stated.

It can take a decade or more to develop a new antibiotic, so it’s vital that we make the best use of existing treatments. One approach is to use combinations of antibiotics for treating the most resistant bacteria. “Dogma suggests that antibiotics ineffective as monotherapy can be effective in combination,” the researchers pointed out, but it’s not clear why a particular combination may only be sporadically effective.

Heteroresistance is a poorly understood mechanism of resistance to some types of antibiotics, in which only a small subpopulation of the bacteria are resistant to a drug. When the antibiotic is used against the whole population the resistant subpopulation can quickly thrive and expand. “We can think of heteroresistance as bacteria that are ‘half resistant’,” Weiss said. “When you take the antibiotic away, the resistant cells go back to being just a small part of the group. That’s why they’re hard to see in the tests that hospitals usually use.”

When testing for antibiotic resistance in a clinical setting, bacteria that exhibit heteroresistance may either be misclassified as susceptible to a particular antibiotic, which can then result in treatment failure, or they may be classified as uniformly resistant. “Clinical antimicrobial susceptibility testing largely relies on liquid media-based diagnostics that assay for growth of the entire population of bacteria in the presence of different antibiotics,” the investigators explained. “This approach only classifies isolate/antibiotic pairs as resistant or susceptible, but cannot differentiate heteroresistance.”

Studies by the Weiss lab and their colleagues now suggest that heteroresistance may be widespread. The researchers evaluated 104 bacterial isolates from a Centers for Disease Control-supported surveillance program in Georgia (the Multi-site Gram-negative Surveillance Initiative) that is tracking multidrug-resistant CRE superbugs. The results of their analyses indicated that 88% of the strains were heteroresistant to at least two antibiotics, and 97.1% were heteroresistant to one of the 16 antibiotics tested.  “Strikingly,” the investigators reported, 23.3% of the isolate/antibiotic interactions that were classified as resistant using clinical testing methods were, in fact, heteroresistant, as were 17.5% of those classified as susceptible by clinical testing methods. “These data indicate that heteroresistance is widespread … and suggest that combination antibiotic regimens targeting multiple heteroresistance may be applicable to a large proportion of CRE.”

But the investigators further showed that this heteroresistance might also provide an opportunity for combination therapy. They found that if bacteria were heteroresistant to two different antibiotics, then the two drugs used in combination more effectively killed the bacteria. The resistant subpopulations were independent, and so didn’t expand together. When the scientists either cultured the bacteria in the presence of one of the antibiotics, or genetically deleted resistance to the drug, the bacteria still exhibited heteroresistance to the other antibiotic of the combination.

The researchers carried out studies using two clinical isolates of multidrug-resistant Klebsiellapneumoniae, designated Nevada-2016 and AR0040. Nevada-2016 was isolated from a woman who had died in a Nevada hospital in 2016, with tests showing that the strain was resistant to 26 different antibiotics, including the last resort drug colistin.

The researchers’ tests showed that despite being designated as pan-resistant, the Nevada bacteria were, in fact, heteroresistant to two antibiotics, fosfomycin and sulfamethoxazole/trimethoprimdespite. When used together the drug combination killed the lab-grown bacteria. A similar approach in live mice showed that animals could survive what should have been a lethal dose of AR0040, when they were treated using a combination of two antibiotics, amikacin and piperacillin/taxobactam, to which the bacterium was found to be heteroresistant.

“Only dual treatment with both amikacin and piperacillin/tazobactam rescued mice from lethal infection, whereas the monotherapies were ineffective,” the researchers stated. “These data highlight that antibiotic combinations targeting multiple heteroresistance can effectively combat isolates deemed pan-resistant by clinical testing and which had been thought to be untreatable.”  The authors stated that their approach highlights “a rational strategy to identify effective combinations that employs existing antibiotics and could be clinically implemented immediately.”

It’s commonly thought that when antibiotic combinations do work its because they act synergistically, with each drug attacking and weakening the organism in a different way. The authors suggest that multiple heteroresistance may more correctly explain why some antibiotic combinations are effective. “Multiple heteroresistance may explain a significant proportion of antibiotic combinations previously identified as synergistic,” the authors wrote.

Weiss commented that drug combinations against multiply heteroresistant bacteria would only be effective if the heteroresistance to each drug didn’t become linked. “Also, we can’t tell beforehand what combination will work—there isn’t any magic combination,” he added. “You have to test the strain. But that isn’t so much different from testing bacterial strains for resistance to individual antibiotics anyway.”

The authors suggested that developing better diagnostics would also prevent misclassification of heteroresistance as susceptibility, in which case monotherapy wouldn’t be effective. Improved diagnostics could also help to identify cases that are heteroresistant, rather than completely resistant, in which case the right combination including the perceived ineffective antibiotic, would be therapeutic. “Rather than designating many instances of heteroresistance as homogenous resistance, and essentially excluding the respective drugs as treatment options, enhanced diagnostics would also highlight antibiotics to which isolates are heteroresistant as potential components of combination regimens … Our data suggest that targeting multiple heteroresistance represents a rational strategy to use clinically approved antibiotics when monotherapy would fail.”



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