Harnessing the Diversity of Burkholderia spp. Prophages for Therapeutic Potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here, we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested phages were tested for lytic activity against the same 32 isolates. Temperate phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that Burkholdera cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Harnessing the diversity of Burkholderia spp. prophages for therapeutic potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested prophages were tested for lytic activity against the same 32 isolates. Lytic phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes, and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that B. cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.