To see how the phages and bacteria might work together, the researchers took advantage of a quirk of the biology of filamentous phages: When these phages infect the bacteria, they do not kill them; rather, the still-living bacteria incorporate the phage DNA into their own DNA and begin churning out lots of viral particles.
The researchers looked at genetic analyses of Pseudomonas bacteria from the lungs of 34 CF patients in Denmark. The patients had had their bacterial DNA sequenced repeatedly over time, allowing the researchers to see whether phage DNA had been persistently incorporated into the bacterial genomes. Patients were more likely to develop consistent phage infections as they got older, supporting the idea that the virus-infected bacteria come to dominate CF patients’ lungs over time. The average age of patients without the phages was 13, while the average age of phage-infected patients was 19.
Burgener and her colleagues also collected sputum samples from 76 people with CF, both adults and children, who were receiving treatment at Stanford. The team tested the sputum for genetic signatures from Pseudomonas and filamentous phages and found that 58 people had Pseudomonas infection. The researchers studied information from the patients’ medical records on lung function, what bacteria had been growing in their lungs over time and other health indicators.
Among the Stanford patients, carrying phage-infected Pseudomonas was more common as patients got older. Phage-infected Pseudomonas bacteria were more likely than bacteria without the virus to be resistant to three antibiotics commonly used to treat CF — aztreonam, amikacin and meropenem — but not to another antibiotic, ciprofloxacin.
“The thing that really stood out was that patients with phage and Pseudomonas had significantly more antibiotic resistance than patients that didn’t have phage,” Burgener said.
How does antibiotic resistance happen?
The researchers previously showed that phage particles glom together into a liquid-crystal structure, a slimy biofilm, which grabs onto antibiotic molecules. In the new study, they tested whether this could prevent antibiotics from diffusing to bacteria. The phage biofilm sequestered aztreonam, amikacin and meropenem away from bacteria, the team showed.
“We think the biofilm is protecting Pseudomonas,” Burgener said. As the biofilm sequesters antibiotics, the bacteria sees sub-therapeutic levels of the drugs, allowing individual drug-resistant bacteria to grow and gradually take over in the lung.
The researchers think the physical properties of the different types of antibiotic molecules — such as whether the drugs have charged or neutral surfaces — may explain why some antibiotics get stuck in the phage biofilm and others do not.
“If we’re able to confirm these results, it may affect how we choose antibiotic therapy for patients who have CF and Pseudomonas,” Burgener said.
It’s shocking how much effect the phages have on the host immune system.
The next step is to understand how CF patients’ bodies respond to the phages, Burgener said, adding, “It’s shocking how much effect the phages have on the host immune system.”
Bollyky recently led another study that suggests it may be possible to vaccinate against the phage.
“Ideally, we’d be able to give a vaccine to CF patients when they’re young,” Burgener said. “Hopefully we can prevent Pseudomonas infection.”
Other Stanford co-authors of the study are graduate students Johanna Sweere, Michelle Bach and Naomi Haddock; former postdoctoral scholar Xiou Cao, PhD; Lu Tian, ScD, associate professor of biomedical data science; and biostatistician Laurence Nedelec, PhD.
Bollyky is a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute at Stanford. Bollyky and Milla are members of the Stanford Maternal & Child Health Research Institute.
Scientists from the University of Montana, Copenhagen University Hospital and the University of Copenhagen also contributed to the research.
The Stanford scientists involved in the research were supported by Stanford’s Maternal & Child Health Research Institute; the Stanford Training Program in Pulmonary, part of a grant from the National Heart, Lung and Blood Institute (grant T32HL129970); Stanford’s Translational Research and Applied Medicine Program; the Ross Mosier Laboratories Gift Fund; the National Institutes of Health (grants R21AI137432 and R01AI12492093); the Cystic Fibrosis Foundation; and the Dr. Ralph and Marian Falk Medical Research Trust Bank of America.