Investigating cooperation and resistance of critically important pathogens

Antibiotic resistance poses a major global health threat, with projections of 8 million deaths in 2050. The World Health Organization has flagged critical bacterial species including the ‘ESKAPEE’ pathogens (<i>Enterococcus faecium</i>, <i>Staphylococcus aureus</i>, <i>Klebsiella pneumoniae</i>, <i>Acinetobacter baumannii</i>, <i>Pseudomonas aeruginosa</i>, <i>Enterobacter </i>spp., and <i>Escherichia coli</i>) and <i>Mycobacterium tuberculosis</i>. Here we investigated mechanisms supporting antibiotic resistance and virulence for these top pathogens via two different approaches.First, we explored how ESKAPEE pathogens cooperate to resist antibiotics and increase virulence traits. <i>S. aureus</i> and <i>K. pneumoniae</i> exhibited the most synergistic relationships with <i>A. baumannii</i>, displaying increased fitness when co-cultured in clinical conditions, and increased virulence in an <i>in vivo</i> model. Broader relationship dynamics of ESKAPEE and <i>A. baumannii </i>pairings demonstrated unique responses for competition assays, cross-feeding assays, and antibiotic resistance profiling.Second, in <i>M. tuberculosis</i>, we identified stepwise genetic changes that drive increasing resistance to first- and last-line antibiotic treatments, rifampicin and linezolid, utilising directed evolution and sequencing resistant communities. Expected resistance mutations were identified (<i>rpoB </i>and <i>rplC</i>), and six low-frequency mutations were uncovered, two of which (<i>ppsD/E</i>) were phenotypically validated as new antibiotic tolerance determinants.This research will inform treatment strategies, diagnosis, and management of bacterial infections, ultimately minimising patient deaths.