Growing antibiotic resistance to a range of infections such as pneumonia, tuberculosis, gonorrhea, and salmonellosis, poses the threat that these infections will become increasingly harder to treat using current antibiotics, resulting in higher cost of treatment, longer hospital stays and higher death rates.

Treatment paradigms using viruses (phages) that infect and kill bacteria without harming humans could help circumvent the looming threat of antibiotic resistance. Phage therapy alone or with antibiotics may cure infections more effectively while reducing the possibility of bacteria developing antibiotic resistance.

“Antibiotic resistance is a major public health issue, and we need to take swift and urgent action. Phage therapy could be an important part of the toolkit, in reducing antibiotic use, and in using them in combination to increase their efficiency,” said Edze Westra, PhD, professor at the department of biosciences at the University of Exeter.

Although phage therapy was first used in 1919 when Félix d’Hérelle, a Parisian microbiologist cured severe dysentery in a 12-year-old boy with a phage cocktail, the rise of antibiotics caused interest in phage therapy to dwindle. Now, growing antibiotic resistance has rekindled interest in phage therapy. However, a major obstacle to the wide adoption of phage therapy is that bacteria also evolve a range to mechanisms that confer rapid resistance against phages.

In a paper published in the journal Cell Host Microbe, titled “Bacteriostatic antibiotics promote CRISPR-Cas adaptive immunity by enabling increased spacer acquisition” on December 20, scientists at the University of Exeter show that exposure to antibiotics that stall bacterial growth without killing them (bacteriostatic antibiotics) slows phage development and provides bacterial cells more time to insert phage-derived spacer sequences into CRISPR arrays in the bacterial genome. This provides the bacterial cells greater CRISPR-Cas adaptive immunity against predatory phages. These finding cast new light on how to best combine antibiotics and phage therapy.

“We found that by changing the type of antibiotics that are used in combination with phage, we can manipulate how bacteria evolve phage resistance, increasing the chances that treatment is effective. These effects should be considered during phage-antibiotic combination therapy, given their important consequences for pathogen virulence,” said Westra, senior author of the paper.

Tatiana Dimitiru, PhD, research fellow at the University of Exeter is lead author of the paper

Tatiana Dimitiru, PhD, research fellow at the University of Exeter and lead author of the paper says, “This study provides fundamental insight into the constraints of CRISPR immune systems in the face of viruses. It was recently discovered that many CRISPR-Cas immune systems are associated to cell responses that make bacteria slow or stop growth upon phage infection, and we speculate that this may be important for cells to trigger an effective immune response.”

In their experiments in the paper, the researchers use an opportunistic human pathogen, Pseudomonas aeruginosa, classified as a priority-one pathogen by the World Health Organization that infects immunocompromised and cystic fibrosis patients and is treated with antibiotics despite emerging multidrug-resistant strains.

To understand how Pseudomonas aeruginosa evolves CRISPR immunity in the presence of antibiotics, the researchers exposed the bacteria to eight different antibiotics and studied how these antibiotics influenced the evolutionary dynamics of the strain P. aeruginosa PA14 in response to its phage DMS3vir. Four of the eight antibiotics that the researchers tested caused a dramatic increase in the levels of CRISPR-based immunity. These antibiotics are all bacteriostatic.

The scientists show the different bacteriostatic antibiotics enhance the evolution of CRISPR immunity in the bacteria by delaying the production of mature phage particles, which allows more time for bacterial genomes to acquire spacer sequences from the phage.

Generalizing their findings to other conditions that decrease the pace of phage replication, the authors show in addition to defective phages and nucleases that cut through phage genomes, the speed of phage replication is key in determining the evolution of CRISPR immunity in bacterial systems. This suggests that the use of bacteriostatic antibiotics can trigger increased levels of CRISPR immunity in human-associated and natural environments.

When a phage infects bacteria, bacteria can rapidly evolve resistance against the phage via their CRISPR-Cas adaptive immune systems or by mutating phage receptors on their surface.

CRISPR immunity depends on inserting a phage-derived sequence into CRISPR arrays in the bacterial genome. By storing pieces of the phage sequence in its genome, the bacteria’s CRISPR system recognizes and attacks the predatory virus in the future.

Alternatively, bacteria can ward off phage infection by losing the receptor on its surface on which the phage docks before entering the bacterial cell. However, this makes the bacteria less virulent, which means they either no longer cause disease or the disease they cause is less severe.

The authors note, “Identifying ways to simultaneously promote spacer acquisition from plasmids and limiting the acquisition of spacers from phage could provide a powerful means to control pathogen abundance, virulence, and their resistance to antibiotics.”

Moving forward, the research team is focusing on understanding how antibiotics and other factors which slow down bacterial growth affect the evolution of CRISPR-Cas immunity in other species and against different types of phages, and studying the consequences of phage-antibiotic combination therapy on bacterial evolution in vivo, says Dimitiru.

The work was funded by the European Research Council.