Christine M. Bojanowski

Pseudomonas aeruginosa (Pa) is one of the predominant bacteria that contributes to chronic respiratory infection, patient morbidity, and mortality in cystic fibrosis (CF) patients. Up to 60% of Pa clinical isolates are hypermutators, caused by deficiency in the DNA mismatch repair (MMR) system, and are associated with increased multidrug resistance and treatment failure. In vitro studies demonstrate a causative relationship between hypermutation and multidrug resistance, yet hypermutators are not currently tested for in the clinical setting as no diagnostic method is currently available. Mutational signatures have been computationally extracted and characterized in human tumors, but to our knowledge this analysis has never been performed in prokaryotes. Here, we evolved MMR-deficient and -proficient Pa in vitro under nine different antibiotic treatments. We sequenced these evolved lineages to test the hypothesis that mutational signature analysis can predict MMR-status and drug resistance. We identified mutation spectra from de novo mutations in the evolved lineages after ten passages and then compared these spectra to known human tumor MMR-deficient mutational signatures. We then sequenced 14 isolates from seven CF patients and performed mutational signature analyses to assess for the presence of MMR-deficient signatures. We observed a strong signature in all evolved MMR-deficient Pa strains of C >T transitions in a NCC and NCG contexts with some 5’ preference to C and G, along with T >C transitions in CTN and GTN contexts. Although many antibiotics are known mutagens, we did not observe treatment-dependent changes in mutational spectra.Mutation spectra consistent with MMR-deficiency were found in four isolates, each of which were also functionally validated as hypermutant via rifampicin reversion assay. These studies suggest that mutational signature analysis may be a valuable tool to identify hypermutator strains of Pa, which could inform treatment regimens and improve management of chronic Pa infections. Kalen M. Hall, B.S., B.A., Informuta: 1|Informuta: Ownership Interest|Informuta: Stocks/Bonds Leonard G. Williams, B.S., Informuta: 1|Informuta: Ownership Interest|Informuta: Stocks/Bonds

Up to 60% of Pseudomonas aeruginosa (Pa) strains isolated from human chronic lung infections lack an active DNA mismatch repair (MMR) system, resulting in a hypermutator phenotype. Hypermutator Pa isolates are associated with enhanced multidrug resistance and treatment failure, and treatments targeting hypermutators are not available. Although hypermutation has been shown to catalyze drug resistance acquisition, it is unclear how treatment of hypermutator Pa influences the development of multidrug resistance. Here, we performed in vitro adaptive evolution of hypermutator Pa under different antibiotic conditions to assess if treatment of hypermutator Pa induces multidrug resistance acquisition via repeated selection for specific resistance-conferring mutations. We serially exposed hypermutator Pa and wild type Pa to four different antibiotics representing three drug classes, and subsequently tested evolved lineages against a panel of six non-treatment antibiotics from varying classes. We observed that hypermutator Pa acquired levels of resistance above clinical breakpoints (defined by Clinical and Laboratory Standards Institute) to all antibiotics tested faster than wild type Pa. Antibiotic treatment of hypermutator Pa selectively induced multidrug resistance acquisition but was irrespective of drug class. Using whole genome sequencing of resistant lineages, we found that treatment-induced multidrug resistance acquisition was determined by shared mechanisms of resistance rather than shared cell target. We subsequently tested combination therapies requiring independent and exclusive mechanisms of resistance and found that combined aztreonam/colistin effectively eliminated multidrug resistance acquisition in hypermutator Pa. Currently, we are testing this combination therapy on 14 Pa isolates (with 4 hypermutator and 10 wild type) from cystic fibrosis patients. Our results shed light on how hypermutator Pa may acquire multidrug resistance within the host and suggest that combination therapies that require independent mechanisms of resistance could potentially reduce emergence of multidrug resistance in hypermutator Pa, providing a viable therapeutic option. Kalen M. Hall, B.S., B.A., Informuta: 1|Informuta: Ownership Interest|Informuta: Stocks/Bonds

Thrombotic thrombocytopenic purpura (TTP), caused by severely reduced activity of the von Willebrand factor-cleaving protease ADAMTS13, is a medical emergency with life-threatening complications and a 90% mortality rate if left untreated. It presents a diagnostic challenge given the multiorgan involvement of the cardiovascular, gastrointestinal, and central nervous systems. Furthermore, the well-known full pentad of fever, hemolytic anemia, bleeding associated with thrombocytopenia, neurological signs, and renal disease is often absent in patients with TTP. We present a 51-year-old male adult with TTP. We utilized the PLASMIC scoring system, which predicts the likelihood of ADAMST13 activity in adults with features of thrombotic microangiopathy and thrombocytopenia with high sensitivity and specificity. We further review the literature supporting the expert statement on ICU management of patients with TTP that plasma exchange (PEX) should be initiated within 6 hours of diagnosis with adjunctive glucocorticoids, rituximab, and caplacizumab. If PEX is unavailable, plasma infusion can be started while the patient awaits transfer to a center with PEX capabilities.