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.

Bicarbonate modulates delafloxacin activity against MDR Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: To investigate the utility of recently approved delafloxacin and other fluoroquinolones against leading MDR bacterial pathogens under physiologically relevant conditions. METHODS: MIC and MBC assays were conducted for MDR strains of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in the standard antibiotic susceptibility testing medium CAMHB, amended Roswell-Park Memorial Institute tissue culture medium (RPMI+) or 20% fresh human whole blood. In vivo correlation of in vitro findings was performed in a murine P. aeruginosa pneumonia model. Mechanistic bases for the findings were explored by altering media conditions and with established fluoroquinolone accumulation assays. RESULTS: Fluoroquinolone MICs were increased in RPMI+ compared with CAMHB for all four MDR pathogens. Specifically, delafloxacin MICs were increased 32-fold versus MDR S. aureus and 8-fold versus MDR P. aeruginosa. MBC assays in 20% human whole blood and a murine MDR P. aeruginosa pneumonia model both confirmed that delafloxacin activity was reduced under physiological conditions. Bicarbonate (HCO3-), a key component of host physiology found in RPMI+ but absent from CAMHB, dictated delafloxacin susceptibility in CAMHB and RPMI+ by impairing its intracellular accumulation. CONCLUSIONS: Standard in vitro antibiotic susceptibility testing conditions overpredicted the effectiveness of delafloxacin against MDR pathogens by failing to capture the role of the biological buffer HCO3- to impair delafloxacin accumulation. This work showcases limitations of our current antibiotic susceptibility testing paradigm and highlights the importance of understanding host microenvironmental conditions that impact true clinical efficacy.

Evolutionary dynamics of pro-inflammatory caspases in primates and rodents.

Caspase-1 and related proteases are key players in inflammation and innate immunity. Here, we characterize the evolutionary history of caspase-1 and its close relatives across 19 primates and 21 rodents, focusing on differences that may cause discrepancies between humans and animal studies. While caspase-1 has been retained in all these taxa, other members of the caspase-1 subfamily (caspase-4, -5, -11, -12, and CARD16, 17, and 18) each have unique evolutionary trajectories. Caspase-4 is found across simian primates, whereas we identified multiple pseudogenization and gene loss events in caspase-5, caspase-11, and the CARDs. Because caspases-4 and -11 are both key players in the non-canonical inflammasome pathway, we expected that these proteins would be likely to evolve rapidly. Instead, we found that these two proteins are largely conserved, whereas caspase-4s close paralog, caspase-5, showed significant indications of positive selection, as did primate caspase-1. Caspase-12 is a non-functional pseudogene in humans. We find this extends across most primates, although many rodents and some primates retain an intact, and likely functional, caspase-12. In mouse laboratory lines, we found that 50% of common strains carry non-synonymous variants that may impact the functions of caspase-11 and -12, and therefore recommend specific strains to be used (and avoided). Finally, unlike rodents, primate caspases have undergone repeated rounds of gene conversion, duplication, and loss leading to a highly dynamic pro-inflammatory caspase repertoire. Thus we uncovered many differences in the evolution of primate and rodent pro-inflammatory caspases, and discuss the potential implications of this history for caspase gene functions.

L. pneumophila resists its self-harming metabolite HGA via secreted factors and collective peroxide scavenging.

IMPORTANCE: Before environmental opportunistic pathogens can infect humans, they must first successfully grow and compete with other microbes in nature, often via secreted antimicrobials. We previously discovered that the bacterium Legionella pneumophila, the causative agent of Legionnaires' disease, can compete with other microbes via a secreted molecule called HGA. Curiously, L. pneumophila strains that produce HGA is not wholly immune to its toxicity, making it a mystery how these bacteria can withstand the "friendly fire" of potentially self-targeting antimicrobials during inter-bacterial battles. Here, we identify several strategies that allow the high-density bacterial populations that secrete HGA to tolerate its effects. Our study clarifies how HGA works. It also points to some explanations of why it is difficult to disinfect L. pneumophila from the built environment and prevent disease outbreaks.