Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens.

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains.

NLRP3 and AIM2 inflammasomes exacerbate the pathogenic Th17 cell response to eggs of the helminth Schistosoma mansoni.

Infection with the helminth Schistosoma mansoni can cause exacerbated morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens, with T helper (Th) 17 cells playing a major role in the development of severe granulomatous hepatic immunopathology. The role of inflammasomes in intensifying disease has been reported; however, neither the types of caspases and inflammasomes involved, nor their impact on the Th17 response are known. Here we show that enhanced egg-induced IL-1ß secretion and pyroptotic cell death required both caspase-1 and caspase-8 as well as NLRP3 and AIM2 inflammasome activation. Schistosome genomic DNA activated AIM2, whereas reactive oxygen species, potassium efflux and cathepsin B, were the major activators of NLRP3. NLRP3 and AIM2 deficiency led to a significant reduction in pathogenic Th17 responses, suggesting their crucial and non-redundant role in promoting inflammation. Additionally, we show that NLRP3- and AIM2-induced IL-1ß suppressed IL-4 and protective Type I IFN (IFN-I) production, which further enhanced inflammation. IFN-I signaling also curbed inflammasome- mediated IL-1ß production suggesting that these two antagonistic pathways shape the severity of disease. Lastly, Gasdermin D (Gsdmd) deficiency resulted in a marked decrease in egg-induced granulomatous inflammation. Our findings establish NLRP3/AIM2-Gsdmd axis as a central inducer of pathogenic Th17 responses which is counteracted by IFN-I pathway in schistosomiasis.

Critical role of growth medium for detecting drug interactions in Gram-negative bacteria that model in vivo responses.

The rise in infections caused by multidrug-resistant (MDR) bacteria has necessitated a variety of clinical approaches, including the use of antibiotic combinations. Here, we tested the hypothesis that drug-drug interactions vary in different media, and determined which in vitro models best predict drug interactions in the lungs. We systematically studied pair-wise antibiotic interactions in three different media, CAMHB, (a rich lab medium standard for antibiotic susceptibility testing), a urine mimetic medium (UMM), and a minimal medium of M9 salts supplemented with glucose and iron (M9Glu) with three Gram-negative ESKAPE pathogens, Acinetobacter baumannii (Ab), Klebsiella pneumoniae (Kp), and Pseudomonas aeruginosa (Pa). There were pronounced differences in responses to antibiotic combinations between the three bacterial species grown in the same medium. However, within species, PaO1 responded to drug combinations similarly when grown in all three different media, whereas Ab17978 and other Ab clinical isolates responded similarly when grown in CAMHB and M9Glu medium. By contrast, drug interactions in Kp43816, and other Kp clinical isolates poorly correlated across different media. To assess whether any of these media were predictive of antibiotic interactions against Kp in the lungs of mice, we tested three antibiotic combination pairs. In vitro measurements in M9Glu, but not rich medium or UMM, predicted in vivo outcomes. This work demonstrates that antibiotic interactions are highly variable across three Gram-negative pathogens and highlights the importance of growth medium by showing a superior correlation between in vitro interactions in a minimal growth medium and in vivo outcomes. IMPORTANCE: Drug-resistant bacterial infections are a growing concern and have only continued to increase during the SARS-CoV-2 pandemic. Though not routinely used for Gram-negative bacteria, drug combinations are sometimes used for serious infections and may become more widely used as the prevalence of extremely drug-resistant organisms increases. To date, reliable methods are not available for identifying beneficial drug combinations for a particular infection. Our study shows variability across strains in how drug interactions are impacted by growth conditions. It also demonstrates that testing drug combinations in tissue-relevant growth conditions for some strains better models what happens during infection and may better inform combination therapy selection.

Implementation of a bundle to improve HIV testing during hospitalization for people who inject drugs.

Background: Increased HIV testing is essential to ending the HIV epidemic. People who inject drugs (PWID) are among the highest risk for HIV infection. Previous research at Tufts Medical Center identified low HIV testing rates in hospitalized PWID. Our research team aimed to identify and overcome barriers to inpatient HIV screening of PWID using implementation science methods. Methods: Stakeholders were engaged to gather perspectives on barriers and facilitators of HIV testing. A PWID care bundle was developed and implemented, which included (1) HIV screening; (2) hepatitis A, B, and C testing and vaccination; (3) medications for opioid use disorder; and (4) naloxone prescription. Strategies from all nine Expert Recommendations for Implementing Change (ERIC) clusters guided the implementation plan. Stakeholder feedback was gathered throughout implementation, and implementation outcomes of acceptability and feasibility were assessed. Results: PWID overall felt comfortable with HIV testing being offered while hospitalized. Clinicians cited that the main barriers to HIV testing were discomfort and confusion around consenting requirements. Many resident physicians surveyed reported that, at times, they forgot HIV testing for PWID. Overall, though, resident physicians felt that the PWID bundle was useful and did not distract from other patient care responsibilities. Conclusions: Engagement of key stakeholders to increase HIV testing in an inpatient setting led to the implementation of a PWID bundle, which was feasible and acceptable. Bundling evidence-informed care elements for inpatient PWID should be investigated further.

People who inject drugs (PWID) are at an increased risk of contracting HIV. HIV testing is a key strategy to stop the spread of HIV. Our study created a bundle of services and tests to offer to all PWID who were admitted to the hospital. The bundle included HIV testing; hepatitis A, B, and C testing and vaccination; medications for opioid use disorder; and prescription for Narcan, a medication that can reverse opioid overdose. We then asked doctors and patients how they felt about the bundle and any barriers and facilitators that they predicted for expanding HIV testing to PWID while admitted to the hospital. Patients were accepting of expanding HIV testing, and resident physicians felt it was important as well and was a manageable addition to their list of responsibilities. However, the most likely part of the bundle to be forgotten was HIV testing. This study lays the groundwork for bundling services for PWID while they are hospitalized. We also highlight areas for future exploration.

The balance between gasdermin D and STING signaling shapes the severity of schistosome immunopathology.

There is significant disease heterogeneity among mouse strains infected with the helminth Schistosoma mansoni. Here, we uncover a unique balance in two critical innate pathways governing the severity of disease. In the low-pathology setting, parasite egg-stimulated dendritic cells (DCs) induce robust interferon (IFN)ß production, which is dependent on the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) cytosolic DNA sensing pathway and results in a Th2 response with suppression of proinflammatory cytokine production and Th17 cell activation. IFNß induces signal transducer and activator of transcription (STAT)1, which suppresses CD209a, a C-type lectin receptor associated with severe disease. In contrast, in the high-pathology setting, enhanced DC expression of the pore-forming protein gasdermin D (Gsdmd) results in reduced expression of cGAS/STING, impaired IFNß, and enhanced pyroptosis. Our findings demonstrate that cGAS/STING signaling represents a unique mechanism inducing protective type I IFN, which is counteracted by Gsdmd.

"They Just Assume That We're All Going to Do the Wrong Thing With It. It's Just Not True": Stakeholder Perspectives About Peripherally Inserted Central Catheters in People Who Inject Drugs.

Background: In the absence of adequate harm reduction opportunities, people who inject drugs (PWID) are at increased risk for serious infections. Infectious diseases guidelines recommend extended periods of intravenous antibiotic treatment through peripherally inserted central catheters (PICCs), but PWID are often deemed unsuitable for this treatment. We conducted semi-structured interviews and focus groups to understand the perspectives and opinions of patients and clinicians on the use of PICCs for PWID. Methods: We approached patients and clinicians (doctors, nurses, PICC nurses, social workers, and case workers) involved in patient care at Tufts Medical Center (Boston, Massachusetts) between August 2019 and April 2020 for semi-structured interviews and focus groups. Results: Eleven of 14 (79%) patients agreed to participate in an in-depth interview, and 5 role-specific clinician focus groups (1 group consisting of infectious diseases, internal medicine, and addiction psychiatry doctors, 2 separate groups of floor nurses, 1 group of PICC nurses, and 1 group of social workers) were completed. Emergent themes included the overall agreement that PICCs improve healthcare, patients' feelings that their stage of recovery from addiction was not taken into consideration, and clinicians' anecdotal negative experiences driving decisions on PICCs. Conclusions: When analyzed together, the experiences of PWID and clinicians shed light on ways the healthcare system can improve the quality of care for PWID hospitalized for infections. Further research is needed to develop a system of person-centered care for PWID that meets the specific needs of patients and improves the relationship between them and the healthcare system.

CD209a Synergizes with Dectin-2 and Mincle to Drive Severe Th17 Cell-Mediated Schistosome Egg-Induced Immunopathology.

The immunopathology caused by schistosome helminths varies greatly in humans and among mouse strains. A severe form of parasite egg-induced hepatic granulomatous inflammation, seen in CBA mice, is driven by Th17 cells stimulated by IL-1ß and IL-23 produced by dendritic cells that express CD209a (SIGNR5), a C-type lectin receptor (CLR) related to human DC-SIGN. Here, we show that CD209a-deficient CBA mice display decreased Th17 responses and are protected from severe immunopathology. In vitro, CD209a augments the egg-induced IL-1ß and IL-23 production initiated by the related CLRs Dectin-2 and Mincle. While Dectin-2 and Mincle trigger an FcRγ-dependent signaling cascade that involves the tyrosine kinase Syk and the trimolecular Card9-Bcl10-Malt1 complex, CD209a promotes the sustained activation of Raf-1. Our findings demonstrate that CD209a drives severe Th17 cell-mediated immunopathology in a helminthic disease based on synergy between DC-SIGN- and Dectin-2-related CLRs.