Since just before the turn of the 20th century, bacteriophage’s antibacterial activity was detected in the waters of the Ganges and Yamuna rivers. English bacteriologist Ernest Hankin had written about the bactericidal effect against cholera of something in the filtered water of these rivers. At the time, he was unaware of viruses that could attack bacteria, and it would be more than two decades later when French-Canadian microbiologist Félix d’Hérelle isolated phage virus from chicken feces and successfully treated an outbreak of chicken typhus—introducing the world to “phage therapy.”

In the 100 years since their first success as a therapy, phages have been used sporadically to treat a host of bacterial infections—gaining more traction in recent years as a therapy to treat drug-resistant bacterial infections. Now, investigators at the National Institutes of Health have just released findings from a recent study using two different bacteriophage viruses individually and then together to successfully treat research mice infected with multidrug-resistant Klebsiella pneumoniae sequence type 258 (ST258). K. pneumoniae ST258 is included on a CDC list of the biggest antibiotic resistance threats in the United States, with high morbidity and mortality rates being associated with the microbe.

Findings from the new study—published recently in mBio through an article titled, “Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption”—note that phages are of great interest today because of a dearth of alternative treatment options for drug-resistant infections. Bacterial resistance has emerged against even the latest drug combinations, leaving some patients with few or no effective treatment options.

Scientists completed a series of studies on research mice infected with ST258. They treated the mice with either phage P1, phage P2, or a combination of the two, and all injected at various times following ST258 infection. The scientists had isolated phages P1 and P2 in 2017 from raw sewage that they screened for viruses that would infect ST258—an indication that phages can be found just about anyplace. Phages P1 and P2 are viruses from the order Caudovirales, which naturally infect bacteria.

“We isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged co-culture in vitro,” the authors wrote. “Single- and double-phage-resistant mutants were isolated from independently evolved replicate co-cultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures.”

Each of the three experimental treatment regimens helped the mice recover from ST258 infection. The scientists noted that the dose of phage provided was less vital to recovery than was the timing of when the dose was received. Mice treated one hour after infection showed the strongest recovery, followed by those treated eight hours after infection and then those treated at 24 hours. Control mice treated with saline all quickly developed severe disease and died.

“All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors,” the authors penned. “Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart).”

The NIH researchers also checked the blood and tissue of phage-treated mice for the presence of ST258 bacteria and found there were significantly fewer bacteria at all time points regardless of the treatment method used, as compared to control mice.

Unfortunately, the scientists also found that ST258 bacteria recovered in the blood and tissue samples of phage-treated mice already had begun developing phage resistance, a finding they are continuing to investigate. The group also is studying how phage therapy results compare between samples of ST258-infected mouse blood and human blood and are examining whether components of human blood can interfere with phage efficacy.

“Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design,” the authors concluded.