Synergy of PMBN and closantel against mcr-1-carrying multidrug- resistant E. coli and Salmonella via membrane permeabilization- driven accumulation and metabolic disruption

In the battle against bacterial infections, scientists have long relied on a small group of powerful drugs known as antibiotics. For decades, these medicines have been the backbone of modern medicine, saving countless lives by killing harmful bacteria or stopping them from multiplying. However, bacteria are clever and adaptable; over time, they have learned to resist these drugs, rendering once-effective treatments useless. This growing resistance is a global crisis, particularly with a specific type of bacteria called Gram-negative bacteria, which have a tough outer shell that makes them naturally difficult to penetrate. When these bacteria acquire additional resistance genes, such as the one that blocks a last-resort antibiotic called colistin, doctors are left with very few options. The search for new solutions has led researchers to look at old drugs in new ways, asking if combining two existing substances might work better than either one alone. One such strategy involves using a helper molecule to break down the bacterial outer shell, allowing a second drug to enter and do its job.

A team of researchers at South China Agricultural University and the Institute of Quality Standards and Testing Technology for Agro Products in China recently explored this exact strategy. They focused on a drug called closantel, which is typically used to treat parasitic worms in animals. While closantel is excellent at killing certain types of bacteria, it fails against Gram-negative bacteria because it cannot get past their tough outer membrane. The researchers wanted to see if they could force closantel inside these resistant bacteria by using a helper molecule called polymyxin B nonapeptide. This helper molecule is a modified version of an antibiotic that can poke holes in the bacterial outer shell without killing the bacteria itself or harming the host. By pairing this helper with closantel, the team aimed to see if they could restore closantel's ability to kill multidrug-resistant strains of E. coli and Salmonella, including those carrying the dangerous mcr-1 resistance gene.

The results of their experiments were striking. When the researchers tested the combination in a laboratory setting, they found that the two substances worked together with remarkable power. Alone, closantel had almost no effect on the bacteria, and the helper molecule alone was also ineffective. But when combined, they acted as a potent weapon, killing the bacteria rapidly. In tests lasting just four hours, the combination reduced the number of living bacteria by a factor of one thousand, and after twenty-four hours, it completely wiped out the bacterial populations in the test tubes. This synergy was consistent across all thirty strains they tested, including the most difficult ones that were resistant to colistin. The researchers also observed that the bacteria did not easily develop resistance to this new combination, even when exposed to it over a month, suggesting that this approach could remain effective over time.

To understand how this combination worked, the scientists looked inside the bacteria. They discovered that the helper molecule successfully breached the outer membrane, acting like a key that unlocked the door for closantel to enter. Once inside, the closantel accumulated to high levels, which triggered a chain reaction of cellular failure. The bacteria's energy supply, measured as ATP, was depleted, leaving them unable to power essential functions. Their ability to pump toxins out of the cell was shut down, and they began to accumulate harmful reactive oxygen species, essentially poisoning themselves from the inside. The researchers also mapped the metabolic changes within the bacteria, finding that the combination disrupted the bacteria's ability to manage energy, balance its internal chemistry, and maintain the integrity of its cell membranes. This total metabolic collapse led to the death of the bacteria.

The team also tested this combination in living mice infected with a resistant strain of E. coli. While the treatment did not save every mouse, it significantly improved their survival rates compared to those that received no treatment or only a single drug. Furthermore, the bacteria found in the organs of the treated mice were far fewer in number. The combination also proved to be safe, causing no damage to red blood cells in the tests, which suggests it would not harm the host. However, the researchers noted that the treatment was less effective in the presence of blood serum, likely because the drug binds to proteins in the blood, reducing the amount available to fight the infection. This indicates that while the biological mechanism is sound, delivering the drug effectively in a living body will require further refinement, such as developing better ways to dissolve and transport the medication.

Ultimately, this study provides a clear demonstration that breaking the outer barrier of resistant bacteria can revive the power of older drugs. By using a non-lethal helper to open the door, the researchers allowed closantel to enter and dismantle the bacteria's internal systems, proving that the barrier to closantel's success was not a lack of targets, but simply the inability to reach them. The findings offer a promising path forward for treating infections that are currently untreatable, highlighting the potential of combining existing drugs to outsmart bacterial resistance. While challenges remain in translating these laboratory successes into human therapies, the work establishes a solid foundation for future research into how we can overcome the growing threat of multidrug-resistant bacteria.