Neutropenic Mice Research Articles

P-1063. SCY-247: A Second-generation IV/Oral Triterpenoid Antifungal with Extensive Tissue Distribution and Pharmacokinetics, and Low Drug-Drug Interaction Potential

Abstract Background SCY-247 is an IV/oral second-generation triterpenoid anti-fungal for systemic fungal infections including multidrug resistant pathogens. Here we present translational data for nonclinical pharmacokinetics (PK) including dose-related tissue disposition in a murine C. glabrata model and correlation to reductions in fungal burden. Further, in vitro CYP450 interactions were evaluated to assess potential for clinical drug-drug interactions (DDIs). Methods The PK of SCY-247 was determined in rats and dogs. Plasma and tissue exposures were compared to reductions in kidney and lung fungal burden in a model of invasive C. glabrata. Neutropenic mice were infected IV with C. glabrata (∼108 yeast cells/mouse) and sacrificed on Day +7. Potential for inhibiting or inducing CYP450 isoforms was assessed in vitro using methods supportive of an FDA Investigational New Drug (IND) Application. Results SCY-247 has low clearance (∼10% liver blood flow) with half-life ≥15 hours following oral and IV administration to rat and dog consistent with once-daily administration in humans. Volume of distribution is ∼3 L/kg supportive of extensive tissue distribution. Exposure increased on repeat administration. Solubility of oral SCY-247 as a salt is >30 mg/mL at gastric pH consistent with rapid dissolution in the stomach and rapid absorption - as demonstrated by an early Tmax (0.5-2hr) following oral administration. In a murine model of C. glabrata infection, SCY-247 demonstrated dose-related increases in exposure and corresponding reduction in fungal burden (right figure panel) with higher distribution in kidney and lung tissue compared to plasma (left figure panel). In vitro, SCY-247 did not induce human CYP450s 1A2, 2B6 or 3A4. The IC50 values for inhibition of human 2C9, 2C19 and 3A isoforms were >20 μM ( >10-fold higher than the anticipated clinical Cmax) supportive of low risk of DDI’s for these isoforms. Conclusion SCY-247 has demonstrated dose- and tissue-related exposure reductions in fungal burden, PK consistent with once daily dosing to humans and low risk for CYP450-related DDIs. Disclosures Steve Wring, PhD, SCYNEXIS, Inc.: Advisor/Consultant Katyna Borroto-Esoda, MS, Scynexis Inc: Advisor/Consultant Nathan P. Wiederhold, PharmD, BioMerieux: Grant/Research Support|F2G: Advisor/Consultant|F2G: Grant/Research Support|Mycovia: Grant/Research Support|Scynexis: Grant/Research Support|Sfunga: Grant/Research Support David A. Angulo, MD, SCYNEXIS, Inc.: Board Member|SCYNEXIS, Inc.: SCYNEXIS, INC Officer and Board Member, SCYNEXIS Patents|SCYNEXIS, Inc.: Stocks/Bonds (Public Company)

P-1864. Early Combination Therapy with Isavuconazole and Immune Checkpoint Inhibitors Improves Infection Outcomes and Alleviates Paralyzed Antifungal Host Immunity in Neutropenic Mice with Invasive Pulmonary Mucormycosis

Abstract Background Invasive pulmonary mucormycosis (IPM) is a severe opportunistic mold infection whose outcome is predominantly host-driven. Preclinical studies and several clinical case reports suggested a benefit of adjunct immunotherapy with immune checkpoint inhibitors (ICIs). However, the mechanisms of IPM-associated pulmonary immune paralysis and its mitigation by ICIs require further study. Timeline of experimental interventions. Abbreviations: CPM = cyclophosphamide, i.p. = intra-peritoneally, ISAV = isavuconazonium sulfate, IT = immunotherapy, PD-L1 = Programmed Death Ligand 1, p.o. = per os (oral gavage). Methods Eight-week-old cyclophosphamide-immunosuppressed BALB/c mice were infected intranasally with 50,000 Rhizopus arrhizus spores. The Mucorales-active triazole isavuconazonium sulfate (ISAV) was initiated on day (d) 3 post-infection (2×64 mg/kg/d by oral gavage). Additionally, mice received 2×0.25 mg/kg anti (α)-PD-L1 or a non-targeting isotype antibody either on d3+5 (early) or d6+8 (late) (Fig. 1). Infection severity was scored using the murine sepsis score (MSS; 0 = healthy – 3 = moribund, 4 = dead). nCounter-based transcriptomics and Ingenuity pathway enrichment analysis were performed on lung tissue homogenates. Murine sepsis scores (MSS, 0 = healthy – 3 = moribund, 4 = dead) of immunosuppressed mice with invasive pulmonary mucormycosis according to the treatment arm. Kruskal-Wallis test with Dunn’s post-test. * p<0.05, *** p<0.001. Abbreviations: ISAV = isavuconazonium sulfate, IT = immunotherapy, ns = not significant, PD-L1 = Programmed Death Ligand 1. Results Untreated neutropenic mice developed severe IPM (median day-10 MSS: 2.9, Fig. 2). Pulmonary transcriptomics at d4 revealed strong innate immune activation and induction of type-1 (Th1) and type-17 (Th17) T-helper-cell responses. However, by d7, proinflammatory signals had markedly decreased and nCounter analysis revealed impaired intercellular crosstalk and T-cell signaling (Fig. 3). Compared to ISAV + isotype (median d10 MSS, 2.0), early and late adjunct α-PD-L1 therapy improved median d10 MSS to 1.0 (p < 0.001) and 1.4 (p = 0.01), respectively (Fig. 2). While ISAV delayed the tipping point from immune activation to full-blown immune paralysis, early adjunct α-PD-L1 therapy elicited significantly stronger dendritic cell and classical (M1) macrophage signaling, activation of key cytokine pathways (e.g., IL-1, IL-2, IL-12), reinvigoration of Th1 and Th17 signaling, and suppression of IPM-induced co-inhibitory checkpoint pathways than both ISAV + isotype and ISAV + late α-PD-L1 (Fig. 3). Enrichment of selected immune-related pathways on day 10 after IPM infection (7 days after initiation of therapy) depending on the treatment arm and comparison with surrogates of immune paralysis in untreated infected mice. All differentially induced pathways were statistically significant (Benjamini-Hochberg adjusted p-values <0.05). Abbreviations: IPM = invasive pulmonary mucormycosis, ISAV = isavuconazonium sulfate, PD-L1 = Programmed Death Ligand 1. Conclusion IPM is associated with rapidly emerging immune paralysis. Combined antifungal and early immunomodulatory (α-PD-L1) therapy was the most effective strategy for early interception of immune paralysis and improvement of morbidity/mortality in our IPM model. Disclosures Sebastian Wurster, MD, MSc, Astellas Pharma: Grant/Research Support|Gilead Sciences: Grant/Research Support Dimitrios P. Kontoyiannis, MD, AbbVie: Advisor/Consultant|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Knight: Advisor/Consultant|Merck: Advisor/Consultant|Scynexis: Advisor/Consultant

P-1221. Benchmark Efficacy for Humanized Cefiderocol Plasma and Pulmonary Epithelial Lining Fluid (ELF) Exposures Against Gram Negative Isolates in Standardized Murine Lung Infection Model

Abstract Background The collaboration for prevention and treatment of MDR bacterial infections (COMBINE) consortium have developed a standardized murine neutropenic lung infection model to align fundamental model elements to reduce inter-laboratory variability. We developed human simulated regimens (HSRs) based on both plasma and ELF exposures for cefiderocol in the COMBINE murine model and herein provide benchmark efficacy against a collection of Klebsiella pneumoniae and Pseudomonas aeruginosa isolates.Table 1.Phenotypic and genotypic information of Klebsiella pneumoniae and Pseudomonas aeruginosa isolates Methods Following the COMBINE protocol for the preclinical murine lung model, neutropenic mice that received uranyl nitrate on day -3 to provide predictable renal impairment were administered 0.05 mL intranasal inoculums of K. pneumoniae (n=9) or P. aeruginosa (n=4) under isoflurane anesthesia. Isolate MICs were determined via BMD. Six mice per isolate were sacrificed 2h after inoculation to determine baseline bacterial burden, and 4-6 received saline control for 24h. Humanized exposures of cefiderocol 2g q8h as 3h infusion in plasma (5, 7.5, 10, 3.5, and 1 mg/kg at 0, 1, 2, 4, and 6h, every 8h) and ELF (3.75, 5, 6.25, 1.75, and 1 mg/kg at 0, 1, 2, 4, and 6h, every 8h) were administered to groups of 6 mice. Efficacy was measured in log10 CFU/lung at 24h compared with baseline bacterial burden.Figure 1.CFU/Lung data in Klebsiella pneumoniae isolates following administration of humanized cefiderocol (2g q8h 3h infusion) plasma and ELF exposures in the COMBINE murine neutropenic lung infection model. Outlier mice determined by Tukey’s Test and displayed as individual dots. Results Cefiderocol MICs and genotypic information are presented in Table 1. The CFU/lung data for K. pneumoniae and P. aeruginosa isolates are displayed in Figures 1 and 2, respectively. The K. pneumoniae isolates required higher log10 CFU/lung starting bacterial burden (mean ± SD, 7.23 ± 0.18) compared with P. aeruginosa (5.68 ± 0.24) to achieve adequate (≥ 1 log10 CFU/lung) growth in 24h controls. With the exception of PSA INT 4-99, the FDC plasma HSR, which over-exposes murine ELF relative to human, achieved greater average CFU reduction than the ELF HSR. Differences in CFU reduction between the matrix HSRs were most pronounced in the 2 isolates with MIC of 16 mg/L at the resistance breakpoint.Figure 2.CFU/Lung data in Pseudomonas aeruginosa isolates following administration of humanized cefiderocol (2g q8h 3h infusion) plasma and ELF exposures in the COMBINE murine neutropenic lung infection model. Conclusion These data provide a comparative benchmark for expected efficacy with humanized cefiderocol exposures of both plasma and ELF when using the COMBINE protocol. Humanizing exposures at the target site of infection may provide better clinical translation when interspecies differences in penetration from plasma exist. Disclosures Andrew J. Fratoni, PharmD, InsightRX: Grant/Research Support Erin Duffy, PhD, CARB-X: Employee David P. Nicolau, PharmD, CARB-X: Grant/Research Support|Innoviva: Grant/Research Support|Innoviva: Honoraria|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria|Venatorx: Grant/Research Support

P-1219. Benchmarks for Humanized Meropenem Plasma and Pulmonary Epithelial Lining Fluid (ELF) Exposures Against Klebsiella pneumoniae in Standardized Murine Lung Infection Model

Abstract Background Inter-laboratory method variability in conducting preclinical murine infection models make data reproducibility and comparisons problematic. The collaboration for prevention and treatment of MDR bacterial infections (COMBINE) consortium have proposed a standardized global protocol to align fundamental elements across laboratories. We developed human simulated regimens (HSRs) based on both plasma and ELF exposures for meropenem in the COMBINE murine neutropenic lung infection model and provide benchmark efficacy against Klebsiella pneumoniae isolates.Table 1.Phenotypic and genotypic information of Klebsiella pneumoniae isolates Methods Following the COMBINE protocol for the preclinical murine lung model, neutropenic mice that received uranyl nitrate on day -3 to provide predictable renal impairment were administered 0.05 mL intranasal inoculums of K. pneumoniae (n=10) under isoflurane anesthesia. Isolate MICs were determined via BMD. Humanized exposures of meropenem 2g q8h as 3h infusion in plasma (65 mg/kg at 0 and 1.25h, 45 mg/kg at 3.5 and 6h, every 8h) and ELF (20 mg/kg at 0h, 50 mg/kg at 1.25h, 35 mg/kg at 3.5h, 20 mg/kg at 4.75h, and 15 mg/kg at 6h, every 8h) were administered to groups of 6 mice. Efficacy was measured in log10 CFU/lung at 24h compared with 0h controls. All isolates except CDC 160 and CDC 848 were tested in duplicate on separate days, with datasets combined and analyzed as one.Figure 1.CFU/Lung data following administration of humanized meropenem (2g q8h 3h infusion) plasma and ELF exposures in the COMBINE murine neutropenic lung infection model. Outlier mice determined by Tukey’s Test and displayed as individual dots. Results Meropenem MICs and genotypic information are presented in Table 1. Mean ± SD initial bacterial burden across all isolates were 7.27 ± 0.21 and 24h net growth in log10 CFU/lung in untreated controls was 1.72 ± 0.34. The CFU/Lung data for each isolate are presented in Figure 1. ≥ 1 log kill was achieved in all 3 non-resistant (MIC ≤ 2 mg/L) isolates and in none of the 7 resistant isolates. Numerical differences in CFU/Lung were seen between the meropenem plasma and ELF HSRs, however there were no categorical differences in achieving ≥ 1 log kill among these 10 isolates. Conclusion Meropenem HSRs based on both plasma and ELF exposure were developed in the COMBINE neutropenic lung infection model and tested against a broad array of K. pneumoniae isolates. These data serve to provide a comparative benchmark for future small molecules in preclinical development against meropenem with these isolates and the standardized protocol. Disclosures Andrew J. Fratoni, PharmD, InsightRX: Grant/Research Support Erin Duffy, PhD, CARB-X: Employee David P. Nicolau, PharmD, CARB-X: Grant/Research Support|Innoviva: Grant/Research Support|Innoviva: Honoraria|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Honoraria|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pfizer: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria|Venatorx: Grant/Research Support

Synergistic effect of repurposed mitomycin C in combination with antibiotics against Aeromonas infection: In vitro and in vivo studies.

Aeromonas infections pose a significant threat associated with high mortality rates. This study investigates the potential of mitomycin C (MMC), an anticancer drug, as a novel antimicrobial agent against Aeromonas infections. We evaluated the minimum inhibitory concentrations (MICs) of MMC and antibiotics against clinical Aeromonas isolates using broth microdilution. Synergistic effects of MMC with antibiotics were determined via time-kill studies. MMC's intracellular killing effects were analyzed using a representative Aeromonas isolate. Efficacy of combined therapies was assessed in a neutropenic mouse model. MMC-induced SOS response was evaluated using cell elongation method, and RNA extraction and quantitative real-time PCR. Combining 1/8⨯ MIC of mitomycin C (MMC) with either 1⨯ or 1/2⨯ MIC of LVX demonstrated significant synergistic effects over 24h in vitro. In a neutropenic mouse model, the combination of MMC (2mg/kg or 1mg/kg) with LVX achieved survival rates of 100% and 80%, respectively, compared to 0% survival with monotherapy. MMC induced marked cell elongation and division inhibition in response to escalating doses. However, the combination therapy's enhancement did not surpass the effects of individual drug treatments. Notably, combination therapy reduced recA activator levels below those observed with either drug alone, suggesting rapid bacterial cell death curtailed further expression of recA and lexA. Alternatively, extensive DNA damage may have overwhelmed bacterial DNA repair mechanisms, rendering them ineffective. These findings suggest that MMC may serve as a potential antimicrobial agent, particularly when used in combination with antibiotics. The integration of MMC with antibiotic therapy offers a promising approach for the treatment of Aeromonas infections and holds potential for future clinical applications.

Bacteriophage treatment is effective against carbapenem-resistant Klebsiella pneumoniae (KPC) in a neutropenic murine model of gastrointestinal translocation and renal infection.

Carbapenemase-producing Klebsiella pneumoniae (KPC) are globally emerging pathogens that cause life-threatening infections. Novel treatment alternatives are urgently needed. We therefore investigated the effectiveness of three novel bacteriophages (Spivey, Pharr, and Soft) in a neutropenic murine model of KPC gastrointestinal colonization, translocation, and disseminated infection. Bacteriophage efficacy was determined by residual bacterial burden of KPC (CFU/g) in kidneys. Parallel studies were conducted of bacteriophage pharmacokinetics and resistance. Treatment of mice with 5 × 109 PFU of phage cocktail via intraperitoneal injection was effective in significantly reducing renal KPC CFU by 100-fold (P < 0.01) when administered every 24 h and 1000-fold (P < 0.01) every 12 h. Moreover, a combination of bacteriophage and ceftazidime-avibactam produced a synergistic effect, resulting in a 105-fold reduction in bacterial burden in cecum and kidney (P < 0.001 in both tissues). Prophylactic administration of bacteriophages via oral gavage did not prevent KPC translocation to the kidneys. Bacteriophage decay determined by linear regression of the ln of mean concentrations demonstrated R2 values in plasma of 0.941, kidney 0.976, and cecum 0.918, with half-lives of t1/2 = 2.5 h. Furthermore, a phage-resistant mutant displayed increased sensitivity to serum killing in vitro, but did not show significant defects in renal infection in vivo. A combination of bacteriophages demonstrated significant efficacy alone and synergy with ceftazidime/avibactam in the treatment of experimental disseminated KPC infection in neutropenic mice.

Pharmacokinetic/pharma-codynamic study of pralurbactam (FL058) combined with meropenem in a neutropenic murine thigh infection model.

Pralurbactam (FL058) is a novel β-lactamase inhibitor with good inhibitory activity on class A, C, and D β-lactamases. This study aimed to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) relationship of pralurbactam/meropenem in a neutropenic murine thigh infection model. After 2-h infection, neutropenic mice was treated with meropenem every 2 h alone or in combination with pralurbactam at different dosing frequencies for 24 h, and the colony count in the thighs was determined before and after treatment. The maximum effect model was fit to the PK/PD relationship to determine the PK/PD index and targets for pralurbactam in combination with meropenem resulting in a static effect and 1-log10 kill. The plasma drug concentration-time data demonstrated that the PK profiles of pralurbactam were consistent with a one-compartment model. Pralurbactam demonstrated a linear PK profile in mice plasma. The percent time of free drug above 1 mg/L (%fT > 1 mg/L) was the PK/PD index that best described the bacterial killing effect of pralurbactam/meropenem over 24 h. When the PK/PD index %fT > 1 mg/L reached 38.4% and 63.6%, pralurbactam/meropenem combination would achieve bacteriostatic effect and 1-log10 reduction against Klebsiella pneumoniae in thigh bioburden, respectively. These PK/PD data derived from mouse thigh infection models will be used to inform the optimal dosing regimen of pralurbactam/meropenem combination in clinical trials.

LC-AMP-I1, a novel venom-derived antimicrobial peptide from the wolf spider Lycosa coelestis.

Antibiotic resistance has become a critical concern in recent years, and antimicrobial peptides may function as innovative antibacterial agents to address this issue. In this work, we identified a novel antimicrobial peptide, LC-AMP-I1, derived from the venom of Lycosa coelestis, demonstrating substantial antibacterial properties and minimal hemolytic activity. LC-AMP-I1 was subjected to additional assessment for antibacterial efficacy, anti-biofilm properties, drug resistance, stability, and cytotoxicity in vitro. It exhibited comparable antibacterial efficacy to melittin against six common clinical multidrug-resistant bacteria, effectively inhibiting biofilm formation and disrupting established biofilms. Additionally, LC-AMP-I1 demonstrated minimal bacterial resistance, excellent stability, negligible mammalian cell toxicity, low hemolytic activity, and appropriate selectivity for both normal and tumor cells. When combined with traditional antibiotics, LC-AMP-I1 exhibited additive or synergistic therapeutic effects. In a neutropenic mouse thigh infection model, LC-AMP-I1 exhibited a therapeutic effect in inhibiting bacterial proliferation in vivo. The mechanistic investigation indicated that LC-AMP-I1 could influence bacterial cell membrane permeability at low concentrations and directly disrupt structure-function at high concentrations. The results of this work indicate that LC-AMP-I1 may function as a viable alternative to traditional antibiotics in addressing multidrug-resistant bacteria.

Development of a pharmacokinetic/pharmacodynamic evaluation model for osteomyelitis and usefulness of tedizolid as an alternative to vancomycin against MRSA osteomyelitis.

This study aimed to develop a suitable osteomyelitis model for pharmacokinetic/pharmacodynamic (PK/PD) evaluation and to investigate the target PK/PD values of vancomycin and tedizolid against methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. An osteomyelitis model was established by implanting an MRSA-exposed sterilized suture in the tibia of normal mice and mice with cyclophosphamide-induced neutropenia. The suitability of the osteomyelitis mouse model for PK/PD evaluation was assessed using vancomycin as an indicator. The target PK/PD values for tedizolid were determined using this model. In neutropenic mice, to achieve a static effect and 1 log10 kill against MRSA, the ratios of the area under the free drug concentration-time curve for 24h to the minimum inhibitory concentration (fAUC24/MIC) of vancomycin were 91.29 and 430.03, respectively, confirming the validity of the osteomyelitis model for PK/PD evaluation. In immunocompetent mice, the target fAUC24/MIC values of tedizolid for achieving a static effect and 1 log10 kill against MRSA were 2.40 and 49.20, respectively. Additionally, only a 0.28 log10 kill was achieved in neutropenic mice with 20 times the human equivalent dose of tedizolid. In patients with restored immunity, tedizolid can potentially be used as an alternative to intravenous vancomycin therapy.

In vivo pharmacodynamic evaluation of the novel nystatin derivative BSG005 in the invasive candidiasis and invasive pulmonary aspergillosis mouse models.

Nystatin, a polyene, is one of the oldest antifungal drugs with wide in vitro potency. BSG005 is a novel, chemically modified, nystatin-like molecule in development for systemic therapy. We evaluated the pharmacokinetic/pharmacodynamic (PK/PD) relationships and target exposures using in vivo invasive pulmonary aspergillosis (IPA) and invasive candidiasis (IC) infection models for BSG005 against common fungal pathogens including Aspergillus fumigatus, Candida albicans, Candida auris, and Candida glabrata. For each species group, three to four strains were selected. Minimum inhibitory concentration (MIC) testing was done by Clinical Laboratory Standards Institute (CLSI) methods. Single-dose kinetics for BSG005 were performed at four dose levels. The immunosuppressed mouse IPA model was used for A. fumigatus studies. For all Candida studies, we utilized the neutropenic disseminated candidiasis model. We used quantitative PCR to enumerate Aspergillus in the lung and colony forming units (CFU) counts for Candida in the kidney. Treatment results were evaluated based on both area under the concentration-time curve (AUC)/MIC and maximum plasma concentration (Cmax)/MIC exposures. The BSG005 MIC was 1 mg/L against all strains. Escalating doses of BSG005 resulted in increased effect and, in general, the dose-response curves within each species were concordant. The median 96-h AUC/MIC associated with net stasis was lowest at 6.08 for C. glabrata. Increasing exposures were needed for same outcome for C. auris at 18.7, C. albicans at 29.3, and A. fumigatus at 102.4. Cmax/MIC targets for the four groups were 0.22, 0.48, 0.60, and 1.41. BSG005 demonstrated potent activity against a variety of fungal pathogens in the neutropenic mouse models. Cmax/MIC PK/PD targets were numerically lower than other polyene studies using the same infection models.

The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo.

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is interest in using bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of i.v. administered bacteriophage in uninfected mice. A single dose of LPS-5, a bacteriophage recently used in human clinical trials to treat drug-resistant Pseudomonas aeruginosa, was administered i.v. to both immunocompetent BALB/c and neutropenic CD1 mice. Phage concentrations were assessed in peripheral blood and spleen at 0.25, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance was only minimally affected by neutropenia. Indeed, the half-lives of phages in blood in BALB/c and CD1 mice were 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that significant inactivation of circulating phages occurs over time. These data indicate that alternative factors, but not neutrophils, inactivate i.v. administered phages.

NLRP1-dependent activation of Gasdermin D in neutrophils controls cutaneous leishmaniasis.

Intracellular pathogens that replicate in host myeloid cells have devised ways to inhibit the cell's killing machinery. Pyroptosis is one of the host strategies used to reduce the pathogen replicating niche and thereby control its expansion. The intracellular Leishmania parasites can survive and use neutrophils as a silent entry niche, favoring subsequent parasite dissemination into the host. Here, we show that Leishmania mexicana induces NLRP1- and caspase-1-dependent Gasdermin D (GSDMD)-mediated pyroptosis in neutrophils, a process critical to control the parasite-induced pathology. In the absence of GSDMD, we observe an increased number of infected dermal neutrophils two days post-infection. Using adoptive neutrophil transfer in neutropenic mice, we show that pyroptosis contributes to the regulation of the neutrophil niche early after infection. The critical role of neutrophil pyroptosis and its positive influence on the regulation of the disease outcome was further demonstrated following infection of mice with neutrophil-specific deletion of GSDMD. Thus, our study establishes neutrophil pyroptosis as a critical regulator of leishmaniasis pathology.

In Vivo Efficacy of Rezafungin, Anidulafungin, Caspofungin, and Micafungin against Four Candida auris Clades in a Neutropenic Mouse Bloodstream Infection Model.

Rezafungin is the first new drug approved to treat candidaemia and invasive candidiasis in more than 10 years. However, data are scant on the in vivo efficacy of rezafungin and the other three approved echinocandins against different Candida auris clades. This study involved 10 isolates representing 4 C. auris clades: South Asian (n = 2), East Asian (n = 2), South African (n = 2), and South American (n = 4, including 2 environmental isolates). In the lethality experiment and fungal tissue burden experiment (kidney, heart, and brain), cyclophosphamide-treated BALB/c male mice were intravenously infected (107 and 8 × 106 colony-forming units [CFU]/mouse, respectively). A 20 mg/kg dose of rezafungin was administered on days 1, 3, and 6. Alternatively, beginning 24 h post-infection, mice received 3 mg/kg of caspofungin, 5 mg/kg of micafungin, or 5 mg/kg of anidulafungin once daily for 6 days. Regardless of isolate and clade, all echinocandin regimens improved survival after 21 days (p = 0.0041 to p < 0.0001). All echinocandins frequently produced >3-log mean CFU/g decreases in the fungal kidney and heart burdens, although some of these decreases were not statistically significant. Rezafungin, regardless of clade, produced 3-5 and 2-4 log CFU/g decreases in the kidney and heart burdens, respectively. Echinocandins did not inhibit fungal growth in the brain. Histopathological examination performed on day 7 showed no fungal cells in the heart and kidneys of rezafungin-treated mice and to a lesser extent, caspofungin-treated mice, regardless of the clinical isolate. All echinocandin-treated mice showed medium and/or large foci of fungal cells in their cerebrum or cerebellum. Regardless of the C. auris clade, rezafungin activity in vivo was comparable to or improved over that of the three previously approved echinocandins.

Adiponectin pathway activation dampens inflammation and enhances alveolar macrophage fungal killing via LC3-associated phagocytosis.

Although innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an adipokine produced mainly in adipose tissue that exerts anti-inflammatory effects in adipose-distal tissues such as the lung. We observed 100% mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN-/- AMs exhibited an inflammatory/M1 phenotype that was associated with decreased fungal killing. Pharmacological stimulation of AMs with AdipoR agonist AdipoRon partially rescued deficient killing in APN-/- AMs that was dependent on both receptors. Finally, APN-enhanced fungal killing was associated with increased activation of the non-canonical LC3 pathway of autophagy. Thus, our study identifies a novel role for APN in LC3-mediated killing of A. fumigatus.

CTC-177, a novel drug-Fc conjugate, shows promise as an immunoprophylactic agent against multidrug-resistant Gram-negative bacterial infections.

The widespread emergence of antibiotic resistance including MDR in Gram-negative bacterial pathogens poses a critical challenge to the current antimicrobial armamentarium. To create a novel drug-Fc conjugate (DFC) that can be delivered at sustained and prolonged levels while simultaneously activating the host immune response to combat MDR Gram-negative infections. The Cloudbreak™ platform was used to develop DFCs consisting of a targeting moiety (TM) (a polymyxin-derived dimer) attached via a non-cleavable linker to an effector moiety (EM) (the Fc domain of human IgG1). In vitro activities of the DFCs were assessed by MIC testing. Neutropenic mouse models of thigh infection, septicaemia and pneumonia were used to evaluate in vivo efficacy. Pharmacokinetics were evaluated in mice and cynomolgus monkeys. A single prophylactic dose of our lead DFC, CTC-177, resulted in significantly decreased bacterial burdens and reduced inflammation comparable to daily treatment with colistin in septicaemia and pneumonia mouse models. Furthermore, CTC-177 prophylaxis was able to restore colistin efficacy in colistin-resistant septicaemia, reducing bacterial burdens beyond the limit of detection. Finally, CTC-177 displayed a long terminal half-life of over 24 and 65 h in mice and cynomolgus monkeys, respectively. These data support the continued development of Cloudbreak™ DFCs as broad-spectrum prophylactic agents against Gram-negative infections.

Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and β-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and β-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Fitness cost of tet(A) type I variant-mediated tigecycline resistance in Klebsiella pneumoniae

ObjectiveThe aim of the present study is to explore the impact of the tet(A) type I variant (tetA-v1) on its fitness effect in Klebsiella pneumoniae. MethodsClinical K. pneumoniae strains were utilized as parental strains to generate strains carrying only the plasmid vector (pBBR1MCS-5) or the tetA-v1 recombinant plasmid (ptetA-v1). Antimicrobial susceptibility testing was conducted to estimate the contribution of tetA-v1 to drug resistance. Plasmid stability was evaluated by serial passage over 10 consecutive days in the absence of tigecycline. Biological fitness was examined through growth curve analysis, in vitro competition assays and a neutropenic mouse thigh infection model. ResultsA 2–4-fold increase in tigecycline MIC was observed following the acquisition of tetA-v1. Without tigecycline treatment, the stability of ptetA-v1 plasmids has been decreasing since day 1. The ptetA-v1 plasmid in Kp89, Kp91, and Kp93 exhibited a decrease of about 20% compared to the pBBR1MCS-5 plasmid. The acquisition of the tetA-v1 gene could inhibit the growth ability of K. pneumoniae strains both in vitro and in vivo. tetA-v1 gene imposed a fitness cost in K. pneumoniae, particularly in the CRKP strain Kp51, with a W value of approximately 0.56. ConclusionThe presence of tetA-v1 is associated with a significant fitness cost in K. pneumoniae in the absence of tigecycline, both in vitro and in vivo.

Efficacy of an oral lipid nanocrystal formulation of amphotericin B (MAT2203) in the neutropenic mouse model of pulmonary mucormycosis.

Invasive mucormycosis (IM) is associated with high mortality and morbidity. MAT2203 is an orally administered lipid nanocrystal formulation of amphotericin B, which has been shown to be safe and effective against other fungal infections. We sought to compare the efficacy of MAT2203 to liposomal amphotericin B (LAMB) treatment in a neutropenic mouse model of IM due to Rhizopus arrhizus var. delemar or Mucor circinelloides f. jenssenii DI15-131. In R. arrhizus var. delemar-infected mice, 15 mg/kg of MAT2203 qd was as effective as 10 mg/kg of LAMB in prolonging median survival time vs placebo (13.5 and 16.5 days for MAT2203 and LAMB, respectively, vs 9 days for placebo) and enhancing overall survival vs placebo-treated mice (40% and 45% for MAT2203 and LAMB, respectively, vs 0% for placebo). A higher dose of 45 mg/kg of MAT2203 was not well tolerated by mice and showed no benefit over placebo. Similar results were obtained with mice infected with M. circinelloides. Furthermore, while both MAT2203 and LAMB treatment resulted in a significant reduction of ~1.0-2.0log and ~2.0-2.5log in Rhizopus delemar or M. circinelloides lung and brain burden vs placebo mice, respectively, LAMB significantly reduced tissue fungal burden in mice infected with R. delemar vs tissues of mice treated with MAT2203. These results support continued investigation and development of MAT2203 as a novel and oral formulation of amphotericin for the treatment of mucormycosis.

Design, Synthesis, and Biological Evaluation of Novel Arylomycins against Multidrug-Resistant Gram-Negative Bacteria.

G0775, an arylomycin-type SPase I inhibitor that is being evaluated in a preclinical study, exhibited potent antibacterial activities against some Gram-negative bacteria but meanwhile suffered defects such as a narrow antibacterial spectrum and poor pharmacokinetic properties. Herein, systematic structural modifications were carried out, including optimization of the macrocyclic skeleton, warheads, and lipophilic regions. The optimization culminated in the discovery of 138f, which showed more potent activity and a broader spectrum against clinically isolated carbapenem-resistant Gram-negative bacteria, especially against Acinetobacter baumannii and Pseudomonas aeruginosa. 162, the free amine of 138f, exhibited an excellent pharmacokinetic profile in rats. In a neutropenic mouse thigh model of infection with multidrug-resistant P. aeruginosa, the potent in vivo antibacterial efficacy of 162 was confirmed and superior to that of G0775 (3.5-log decrease vs 1.1-log decrease in colony-forming unit (CFU)). These results support 162 as a potential antimicrobial agent for further research.

Role of the osaA Gene in Aspergillus fumigatus Development, Secondary Metabolism and Virulence.

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. The deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for the expression of the conidiation regulatory genes brlA, abaA, and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, the deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both neutropenic and corticosteroid-immunosuppressed mouse models.