Description
Antibiotic resistance represents one of the top public health threats of the 21st century. Pseudomonas aeruginosa is a Gram-negative ESKAPE pathogen that commonly causes antibiotic-resistant infections in patients with compromised immunity, such as those with respiratory diseases like chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). One recently revived antimicrobial treatment for respiratory infections is phage therapy, which employs viruses known as bacteriophages to eradicate bacterial infections. However, atmospheric conditions in the body at the site of bacterial infection are often oxygen-deficient or hypoxic, which may alter the efficacy of phage treatment. This project aimed to determine the role that oxygen availability plays in phage-bacteria dynamics and the development of phage resistance, using P. aeruginosa in an in vitro model of hypoxia. Phage infection was carried out under typical atmospheric conditions (21% O2 / 0.4 % O2) and hypoxia (14% O2 / 5.6 % O2). Kinetics experiments revealed that hypoxia slows bacterial growth, which correlated with a delay in phage resistance and decrease in final density of phage-resistant outgrowth. Despite this delay, in hypoxia the frequency of events conferring phage resistance was increased. This was observed as a decrease in the frequency of phage clearing. Under normoxia, phage clearing occurred regularly and increased with phage dose. However, under hypoxia, all cultures developed phage resistance. Additionally, under normoxia, certain phage-resistant mutants produced red pigment. Pigment production did not occur in phage-resistant mutants grown under hypoxia. Sequencing of normoxic and hypoxic phage-resistant isolates revealed that mutations in the LPS synthesis genes galU and ssg commonly evolved in both conditions. However, in normoxia, large chromosomal deletions (LCDs) in galU were the most prevalent mutation. LCDs were also found to cause red pigmentation due to excision of an additional gene, hmgA. LCDs also excised a host of regulatory genes. In hypoxia, LCDs were far less frequent, with phage infection favoring evolution of single nucleotide polymorphisms (SNPs) in galU. Further investigation is required of the relationship between oxygen availability and phage resistance in order to appropriately utilize phage therapy to treat respiratory bacterial infections.
