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2390. Avibactam Sensitizes NDM Klebsiella pneumoniae to Innate Immune Killing by Human Cathelicidin LL-37, Serum, Neutrophils, and Platelets

Abstract Background Avibactam (AVI) is a broad-spectrum intravenous non-β-lactam/β-lactamase inhibitor with no reported activity against metallo-β-lactamases such as New Delhi metallo-β-lactamases (NDM). Structural similarities between β-lactamases and bacterial penicillin-binding proteins (PBPs) have led investigators to explore and confirm the hypothesis that AVI may interact with PBPs of several Gram-negative and -positive bacterial species. Potent synergy has also been observed between AVI and peptide antibiotics such as polymyxin B. We hypothesized that sub-bacteriostatic concentrations of AVI may bind PBPs to weaken cell wall integrity and enhance lysis by the membrane attack complex of complement and by endogenous cationic antimicrobial peptides (AMPs) such as human cathelicidin LL-37. Sensitization to endogenous AMPs could improve killing by neutrophils and platelets that release these effectors upon degranulation. Methods Using NDM K. pneumoniae (NDM-KP) as a model, we performed LL-37 kill curves and killing assays with human serum, neutrophils and platelets in the presence or absence of AVI 4 μg/mL against NDM-KP. Results AVI alone lacked in vitro activity against NDM-KP. Addition of AVI to a physiological achievable concentration of LL-37 (2 mM) was bactericidal and resulted in an 8-log10 reduction (below detection limit) in recoverable NDM-KP CFU at 6 and 24 h; no bactericidal activity was seen in bacteria treated with LL-37 or AVI alone (P < 0.0001). AVI pretreatment dramatically sensitized NDM-KP to neutrophil and platelet killing (P < 0.0001 and P < 0.01, respectively). AVI also sensitized NDM-KP to 20% human serum, resulting in 8-log10 reduction in recoverable NDM-KP CFU within 6 h (P < 0.0001), an effect abrogated by heat treatment to inactivate complement. Conclusion AVI demonstrates potent synergy with peptide antibiotics and the innate immune system in vitro. Since AVI alone has scant direct antimicrobial activity and no direct inhibitory effect on metallo-β-lactamases, it is less likely to increase selective pressures toward antibiotic resistance. The use of AVI in combination with other antibiotics against drug-resistant bacterial pathogens warrants further study. Disclosures G. Sakoulas, Allergan: Consultant and Speaker, Consulting fee and Speaker honorarium. Sunovion: Speaker, Speaker honorarium. The Medicines Company: Speaker, Consulting fee. Paratek Pharmaceuticals: Consultant, Consulting fee. Cidara Therapeutics: Scientific Advisor, None. Arsanis Pharmaceuticals: Scientific Advisor, None.

Methods. Mice received either humanized exposures of cefiderocol equivalent to the clinical dose [2g q8h 3h inf.] or cefepime (FEP) reflective of a 2g q8h 3h inf or vehicle. Efficacy was determined as the change in log CFU at 24, 48 and 72h compared with 0 hours controls. MICs were determined on organisms recovered from both the control and treatment animals.
Results. In AB, PSA and Enterobacteriaceae (EB) displaying MICs 0.5-8 (n = 9), infected mice given cefiderocol showed reductions of 0.5-3 log CFU at 72 hours. The killing profile observed among these 9 isolates followed a similar trend, demonstrating an initial reduction in bacterial burden at 24 hours which was sustained at 48 hours and 72 hours. As expected based on the PD profile of cefiderocol, no killing was seen with the AB isolate (MIC = 16). While cefiderocol exposure resulted in the killing of the FEP-resistant phenotype of the EB, mice receiving FEP displayed growth similar to controls. Infection with the remaining 2 organisms (EC 462, MIC = 1; KP 531, MIC = 4) resulted in a cumulative increase in bacterial burden over the study duration resulting in 1-2 logs growth following cefiderocol exposure over 72 hours. Retest MICs revealed an increase (≥2 dilutions) compared with control in only 1 animal (1/54 samples or 1.8%) observed in EC 462 at 72 hours. Additional samples from this group (2/3) remained unchanged throughout the study duration. Importantly, the retest MIC for this sample did not exceed the MIC of 4 mg/L.

Conclusion.
These data show that for isolates demonstrating kill at 24 hours, cefiderocol efficacy was unchanged over the 72 hours treatment period. Despite the MDR profile of the pathogens tested their phenotypic profile remained largely unchanged and adaptive resistance during therapy was not observed.
Disclosures. M. Tsuji, Shionogi & Co., Ltd.: Employee, Salary. Y. Yamano, Shionogi & Co., Ltd.: Employee, Salary. Background. Rezafungin (RZF), a novel, once-weekly echinocandin for treatment and prophylaxis of invasive fungal infections, successfully met safety and efficacy endpoints in Phase 2 and is advancing to Phase 3 studies. RZF is the first echinocandin to undergo a definitive QT evaluation.

Effect of Rezafungin on QT Interval in Healthy Subjects
Methods. This Phase 1, single-center, randomized, double-blind, comparative study evaluated the effects of RZF on the QTcF (corrected using Fridericia's formula) interval), heart rate, and other cardiac parameters. There were three dose groups, RZF (600 mg or 1,400 mg IV), IV placebo, and oral moxifloxacin (positive control). The 600 mg (therapeutic) and 1400 mg (supratherapeutic) doses were selected to achieve exposures approximating those after multiple doses of the highest dosage regimen assessed in the Phase 2 study (400 mg once weekly) and exposures ~2.5-fold higher, respectively. The primary endpoint was based on an analysis of change of QTcF from Baseline (ΔQTcF) as a function of RZF plasma concentration, to derive the estimated mean placebo-adjusted change of QTcF from Baseline (ΔΔQTcF) for the RZF dose groups at the geometric mean Cmax for each dose level. The outcome was defined by a comparison of the upper bounds of the 2-sided 90% CIs within 10 ms.
Results. 60 subjects were enrolled and completed the study. Demographics included: sex (43.3% male) and age (median age 34.0 years; ranging from 20 to 51 years) approximately evenly distributed by treatment. A linear regression model best fit the data, as shown in Figure 1. From this model, the estimated mean ΔΔQTcF at the Cmax for both of the RZF doses had upper bounds <10 ms. The mean ΔΔQTcF at each time point by dose, showed all 1-sided 95% upper bounds to be <10 ms, thus supporting the conclusion of the primary analysis. Assay sensitivity was established for moxifloxacin. No clinically significant effects on any of the cardiac parameters tested (RR, QRS, HR) were observed. RZF was generally well tolerated. All adverse events (AE) were mild to moderate in severity with no discontinuations due to AEs.
Conclusion. Rezafungin, in single IV doses up to 1,400 mg, does not prolong the QT interval. This finding supports the clinical safety and continued development of RZF.
Poster Abstracts • OFID 2018:5 (Suppl 1) • S713 Background. Avibactam (AVI) is a broad-spectrum intravenous non-β-lactam/ β-lactamase inhibitor with no reported activity against metallo-β-lactamases such as New Delhi metallo-β-lactamases (NDM). Structural similarities between β-lactamases and bacterial penicillin-binding proteins (PBPs) have led investigators to explore and confirm the hypothesis that AVI may interact with PBPs of several Gram-negative and -positive bacterial species. Potent synergy has also been observed between AVI and peptide antibiotics such as polymyxin B. We hypothesized that sub-bacteriostatic concentrations of AVI may bind PBPs to weaken cell wall integrity and enhance lysis by the membrane attack complex of complement and by endogenous cationic antimicrobial peptides (AMPs) such as human cathelicidin LL-37. Sensitization to endogenous AMPs could improve killing by neutrophils and platelets that release these effectors upon degranulation.
Methods. Using NDM K. pneumoniae (NDM-KP) as a model, we performed LL-37 kill curves and killing assays with human serum, neutrophils and platelets in the presence or absence of AVI 4 μg/mL against NDM-KP.
Results. AVI alone lacked in vitro activity against NDM-KP. Addition of AVI to a physiological achievable concentration of LL-37 (2 mM) was bactericidal and resulted in an 8-log 10 reduction (below detection limit) in recoverable NDM-KP CFU at 6 and 24 h; no bactericidal activity was seen in bacteria treated with LL-37 or AVI alone (P < 0.0001). AVI pretreatment dramatically sensitized NDM-KP to neutrophil and platelet killing (P < 0.0001 and P < 0.01, respectively). AVI also sensitized NDM-KP to 20% human serum, resulting in 8-log 10 reduction in recoverable NDM-KP CFU within 6 h (P < 0.0001), an effect abrogated by heat treatment to inactivate complement.
Conclusion. AVI demonstrates potent synergy with peptide antibiotics and the innate immune system in vitro. Since AVI alone has scant direct antimicrobial activity and no direct inhibitory effect on metallo-β-lactamases, it is less likely to increase selective pressures toward antibiotic resistance. The use of AVI in combination with other antibiotics against drug-resistant bacterial pathogens warrants further study.
Disclosures. G. Sakoulas, Allergan: Consultant and Speaker, Consulting fee and Speaker honorarium. Sunovion: Speaker, Speaker honorarium. The Medicines Company: Speaker, Consulting fee. Paratek Pharmaceuticals: Consultant, Consulting fee. Cidara Therapeutics: Scientific Advisor, None. Arsanis Pharmaceuticals: Scientific Advisor, None. Background. Biofilms are sophisticated communities of matrix-encased and surface-attached bacteria that exhibit a distinct and specific resistant/tolerant phenotype to almost all antibacterial agents, with activity reduced 10-to 1,000-fold. Interestingly, this augmented resistance rapidly reverts when bacteria detach from the biofilm and return to a planktonic state. However, in this in vitro pharmacokinetic and pharmacodynamic (PK/PD) model we are able to expose biofilms to shear rates that are consistent with human interface and mimic antibiotic penetration and diffusion pathways from serum antibiotic concentration in humans.

Liposomal Vancomycin and Cefazolin Combinations for S. aureus Biofilms
Methods. Methicillin-susceptible ATCC 29213 and MRSA 494 strains were evaluated. Initial susceptibility tests were performed by broth microdilution method. Time kill studies were performed to identify synergy patterns for liposomal and commercial antibiotics. Biofilm eradication was investigated using antibiotics vancomycin (VAN) (commercial) vs. liposomal VAN (VAN-L) (Patent#17-1460) and also combination of VAN-cefazolin (commercial) vs. liposomal vancomycin and liposomal cefazolin (CFZ-L) (Patent# 17-1460) in biofilms for strain MRSA 494. Biofilms were generated overnight using the BioFlux Microfluidic system (Fluxion BioSciences) at constant and continuous shear rates to optimize biofilm attachment and creation. Perfusion of antibiotic solutions (free peak concentration) was applied over a 24 h time period. Time lapse pictures were recorded to determine antibiotic biofilm eradication rates over 18h of incubation and pictures were analyzed using Bioflux Montage software.
Results. MIC values demonstrated a 2-fold reduction for liposomal vancomycin vs. commercial vancomycin. Also, combination of liposomal VAN MIC in presence of CFZ-L showed a 15.87-fold reduction in comparison to commercial VAN for 494. Overall, our biofilm results demonstrated a 43.6% improved eradication using VAN-L and CFZ-L combination in comparison to commercial VAN-CFZ combination. We also observed 5.7% improved eradication using VAN-L vs. commercial VAN. Conclusion. Liposomal form of VAN and CFZ combinations are a promising approach to improved efficacy and reduced VAN  Background. Urinary tract infection (UTI) is one of the most common infectious diagnoses and in 2007 accounted for 10.5 million primary care visits in the US Advancing age and comorbidities, such as chronic kidney disease (CKD) and diabetes, affect antimicrobial prescribing habits. Sulfamethoxazole/trimethoprim (SMX-TMP), nitrofurantoin, and fosfomycin are first-line recommendations for uncomplicated cystitis. In an aging male population with potential allergies or contraindications to the above, fosfomycin is a potential option for treatment.
Methods. A retrospective chart review of fosfomycin prescribing habits at a large VA academic medical center. Patients were selected based on fosfomycin prescription in both inpatient and outpatient settings from January 1, 2004 to December 5, 2017. Data reviewed included indication, organism(s), susceptibility, duration of treatment, CKD, and clinical success. Treatment success was defined as no representation with UTI symptoms for 30 days.
Results. 117 cases of UTI in which fosfomycin was used were identified with a median patient age of 70 years old and 90% male. Twenty-five were uncomplicated cystitis, 49 complicated cystitis, and 34 catheter associated infections. Treatment success was obtained in 92% of the uncomplicated cystitis cases, 76% in complicated cystitis cases, and 67% in catheter associated UTIs. In half of all the cases an ESBL bacterium was isolated and 79% were successfully treated with fosfomycin. The most common pathogen identified was E. coli 58/118 (49%), followed by Klebsiella 25/118 (21%).
Conclusion. Fosfomycin is an antibiotic recommended for simple cystitis due to its safety profile, less collateral damage (gut flora disturbance), and low resistance as currently known. This review displays the largest ESBL cohort identified in the literature and uniquely used in a predominant male population. These findings suggest that ESBL producing bacteria can be treated successfully with fosfomycin in a male population as well as uncomplicated cystitis. However, caution should be used with catheterized patients as treatment was less effective regardless of isolated bacteria.
Disclosures. All authors: No reported disclosures.