Stability of Enveloped and Nonenveloped Viruses in a Stable Gelatin Liquid Formulation

The thermal stability of relevant viruses in gelatin liquid formulations for medical research and application is poorly understood. Bovine herpesvirus (BHV) was used as a model virus to examine the molecular weight (MW), concentration and gelatin type and to optimize virus stability in liquid formulations at 25 °C and 4 °C. Using the model virus stable liquid formulation, the stability of multiple enveloped and nonenveloped RNA and DNA viruses, including parainfluenza virus (PIV), reovirus (RV), BHV, and adenovirus (AdV), was monitored over up to a 30-week storage period. The BHV model virus was considered stable after 3 weeks in hydrolyzed gelatin (MW: 4000) with a 0.8 LRV (log10 reduction value) at 25 °C or a 0.2 LRV at 4 °C, compared to the stabilities observed in higher MW gelatin (60000 and 160000) with an LRV above 1. Based on the gelatin type, BHV in B-type gelatin samples were unexpectantly more stable than in A-type gelatin sample. All four viruses exhibited stability at 4 °C for at least 8 weeks, BHV or AdV remained stable for over 30 weeks of storage, and at 25 °C, AdV and RV remained stable for 8 weeks. The results demonstrated that 5% hydrolyzed gelatin can act as a relevant stabilizer for the thermal stability of viruses in medical research and application.

1035. Manufacturing Processes of SER-109, a Purified Investigational Microbiome Therapeutic, Reduce Risk of Transmission of Emerging and Undetected Infections in Donor Stool

Background Fecal microbiota transplantation (FMT) is vulnerable to emerging pathogens due to reliance on donor screening for risk mitigation. These concerns were highlighted by dual FDA safety alerts regarding FMT transmission of bacterial pathogens, which were recognized in hindsight only after hospitalizations and deaths. The FDA also warned of potential risk of SARS-CoV-2 transmission, leading to quarantine of FMT in March 2020, two months after COVID-19 was reported on US soil. Conversely, our development program for SER-109, an oral investigational microbiome therapeutic, was prospectively designed to inactivate organisms of concern, while purifying the hardy Firmicutes spores. We evaluated whether the manufacturing processes for SER-109 inactivate model organisms, including a coronavirus with gastrointestinal tropism, and a representative Gram-negative bacterium. Methods Model organisms were selected based on biologic suitability, detectability, and laboratory safety. Porcine Epidemic Diarrhea Virus (PEDV, a coronavirus) was selected to model SARS-CoV-2. Quantitation used a Vero cell tissue culture infectious dose (TCID50) assay. For E. coli, a rifampicin-tolerant Salmonella enterica was selected and quantified with MacConkey lactose agar plus rifampicin. Spiking experiments into representative fecal suspensions were completed to measure inactivation of model organisms. Log-reduction factors (LRF) were calculated based on the drop in organism titer during inactivation. Hold controls in non-ethanolic test matrices were used to confirm specificity of the ethanol inactivation. Results In 70% v/v ethanol, PEDV was inactivated by more than 4.2 log10 (to limit of detection, LOD) within 4 minutes (Fig1). In 50% v/v ethanol, S. enterica was inactivated by more than 6.5 log10 (to LOD) within 30 seconds (Fig2). Figure 1. Inactivation of Porcine Epidemic Diarrhea Virus (PEDV), log10 reduction factor (LRF) versus time Average of two experiments shown. Also shown is the maximum achievable inactivation based on the limit of detection (LOD). Figure 2. Inactivation of S. enterica, log10 reduction factor (LRF) versus time. Average of three experiments with error bars represent 95% CI. Also shown is the maximum achievable inactivation based on the limit of detection (LOD). Conclusion These experiments demonstrate substantial inactivation of the model organisms and support the potential benefit of SER-109 manufacturing process to mitigate risks of undetected or emerging pathogens for which reliable screening is limited. Ethanol exposure leads to a purified investigational product of beneficial Firmicutes spores while affording a safety net beyond donor screening alone. Disclosures Christopher McChalicher, n/a, Seres Therapeutics (Employee, Shareholder) Ahmad Abdulaziz, MS, Seres Therapeutics Inc. (Employee, Shareholder) Elizabeth Halvorsen, PhD, Seres Therapeutics (Employee, Shareholder) Mary-Jane Lombardo, PhD, Seres Therapeutics (Employee, Shareholder) Jonathan Winkler, PhD, Seres Therapeutics (Employee, Shareholder) Barbara McGovern, MD, Seres Therapeutics (Employee, Shareholder) Gregory McKenzie, PhD, Prolacta Bioscience (Employee) David Ege, PhD, Merck & Co., Inc. (Shareholder)Seres Therapeutics (Employee, Shareholder) John Aunins, PhD, Seres Therapeutics, Inc. (Employee)

1103. Minocycline (MIN) Pharmacodynamics (PD) against Stenotrophomonas maltophilia (STM) in a Neutropenic Murine Thigh Infection Model

Background Antibiotic treatment options for serious STM infections are limited. MIN displays in vitro activity against STM; however, limited data supports optimal dosing for STM. Herein, we employed the murine neutropenic thigh infection model to assess MIN PD against STM. Methods Four clinical STM isolates with MIN MICs 0.25 – 1 mg/L were included. Both thighs of neutropenic ICR mice were inoculated with bacterial suspensions of 107 colony forming units (CFU)/mL. Mice received uranyl nitrate on Day -3 to provide predictable renal impairment. Two hours after inoculation, MIN or control was administered subcutaneously. Pharmacokinetic (PK) studies of 2.5, 25, 50, and 100 mg/kg were conducted. Previously reported protein binding of 78.1% was used to define free exposure. Dose ranging studies were conducted on all STM to assess in vivo activity over a range of MIN exposures. MIN total daily doses (TDD) of 10, 20, and 50 mg/kg were fractionated q24h, q12h, and q6h against a single STM to determine the PD index best correlated with reductions in CFU/mL. Efficacy was measured in log10CFU/thigh at 24h compared with 0h controls. Composite CFU data were fitted to an Emax model to determine the fAUC/MIC exposure for stasis and 1 log10 reduction. Results MIN PK was linear up to 50 mg/kg and well described by a 1 compartment model with first order absorption and elimination. Mean PK parameters across the linear range were: Vd, 2.97 L/kg; K01, 10.62 1/h; and K10, 0.35 1/h. Mean ± SD bacterial burden at 0h across all isolates was 6.17±0.20 log10CFU/thigh. In 24h controls, bacterial growth was 7.90±0.85 log10CFU/thigh. A dose response was observed across all isolates using TDD of 2-300 mg/kg. PD indices correlated with CFU reductions as follows: fAUC/MIC (R2=0.613), fCmax/MIC (R2=0.590), and %fT >MIC (R2=0.504). The fAUC/MIC needed for stasis and 1 log10 reduction at 24h was 9.6 and 23.6, respectively. Conclusion These are the first data describing MIN PD against STM. Against these STM, MIN fAUC/MIC was the PD index best correlated with CFU reductions. The exposure thresholds defined in this study will be useful in designing optimal MIN dosing regimens for treating STM infections and re-assessment of the current susceptibility breakpoint. The study was funded under FDA Contract 75F40120C00171. Disclosures David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase (Other Financial or Material Support, I have been a consultant, speakers bureau member, or have received research funding from the above listed companies.) Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)BioMérieux (Consultant, Research Grant or Support, Speaker’s Bureau)Contrafect (Scientific Research Study Investigator)GSK (Consultant)Merck (Research Grant or Support)Paratek (Speaker’s Bureau)Roche Diagnostics (Research Grant or Support)Shionogi (Research Grant or Support)Summit (Scientific Research Study Investigator)

Levofloxacin pharmacodynamics against Stenotrophomonas maltophilia in a neutropenic murine thigh infection model: implications for susceptibility breakpoint revision

Background Levofloxacin displays in vitro activity against Stenotrophomonas maltophilia (STM); however, current susceptibility breakpoints are supported by limited data. We employed the murine neutropenic thigh infection model to assess levofloxacin pharmacodynamics against STM. Methods Twenty-six clinical STM were studied using the neutropenic murine thigh infection model. Human simulated regimens (HSR) of levofloxacin 750 mg q24h were administered over 24 h. Efficacy was measured as the change in log10 cfu/thigh at 24 h compared with 0 h. Composite cfu data were fitted to an Emax model to determine the fAUC/MIC needed for stasis and 1 log10 reduction at 24 h. Monte Carlo simulation was performed to determine PTA. Results Levofloxacin MICs ranged from 0.5–8 mg/L. Mean bacterial burden at 0 h was 6.21 ± 0.20 log10 cfu/thigh. In the 24 h controls, bacterial growth was 1.64 ± 0.66 log10 cfu/thigh. In isolates with levofloxacin MICs ≤1, 2 and ≥4 mg/L, changes in bacterial density following levofloxacin HSR were −1.66 ± 0.89, 0.13 ± 0.97 and 1.54 ± 0.43 log10 cfu/thigh, respectively. The Emax model demonstrated strong agreement between fAUC/MIC and change in bacterial density (R2 = 0.82). The fAUC/MIC exposure needed for stasis and 1 log10 reduction was 39.9 and 54.9, respectively. PTAs for the 1 log10 reduction threshold were 95.8, 72.2, and 26.6% at MICs of 0.5, 1 and 2 mg/L, respectively. Conclusions These are the first data to describe fAUC/MIC thresholds predictive of cfu reductions for levofloxacin against STM. Due to poor in vivo efficacy and PTA at MICs ≥2 mg/L, reassessment of the current susceptibility breakpoint is warranted.

Proof of Concept: The Effectiveness of Disinfectant Tunnel as Potential Measure against COVID-19

Disinfectant tunnels have attracted attention as a potential measure to prevent the spread of COVID-19, but their safety and effectiveness are questionable. Disinfectants such as sodium hypochlorite were used, yet no scientific evidence is available on its effectiveness to eliminate SARS-CoV-2 on the human body through spraying, although this chemical is effective in the elimination of the virus on inanimate surfaces. Since safety issues are of importance, countries have halted the operation of these tunnels. Available literature has suggested several effective disinfectants against SARS-CoV-2, including iodine-based solution, such as povidone-iodine (PVP-I). This report describes the evaluation of the bactericidal activity of PVP-I in comparison to sodium hypochlorite, both in vitro and following spraying under a model tunnel. Two bacteria strains (Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 12600) were used as model microorganisms. The spraying pattern and droplets distribution from the tunnel are also being described and were correlated with the effectiveness of the disinfectant droplets to eliminate the model bacteria. Results showed that 0.5 % v/v PVP-I and 0.5% v/v sodium hypochlorite are bactericidal (> 5 log10 reduction) in vitro. However, sprayed disinfectants did not show similar activity. Bacterial growth was seen in all cloth samples for 0.5% v/v PVP-I and all cloth samples except right shoulder for 0.5 % v/v sodium hypochlorite. Hence, the design of any disinfectant tunnel is important, and with an effective disinfectant to justify its efficacy.

Application of chitosan microparticles against human norovirus

Human norovirus (HuNoV) is the leading causative agent of foodborne outbreaks and is associated with the second most prevalent cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CM) against HuNoV GII.4 Sydney and its cultivable surrogate, Tulane virus (TuV), in suspensions mimicking fecally-contaminated water. CM was prepared by crosslinking chitosan molecules with sodium sulfate, and then its anti-noroviral activity was assessed using infectivity assay on TuV and RT-qPCR on TuV and HuNoV. A 3% CM suspension in PBS (pH 7.2) showed binding to TuV particles but with a negligible impact on virus infectivity (p>0.05). TuV and HuNoV suspended in fecal suspensions showed a 1.5-log10 reduction in genomic copies per ml following a 10-min contact time (p<0.05). Despite the negligible impact on viral infectivity, CM moderately binds to virus particles and helps purify environmental water by removing infectious virus particles. In this study, TuV served as a suitable surrogate for HuNoV by showing a similar log10 reduction in fecal suspension. Overall, the outcomes of thisresearch highlight the potential application of CM as a novel, natural treatment to minimize the spread of water-transmitted viral pathogens.

Activity of N-Chlorotaurine against Long-Term Biofilms of Bacteria and Yeasts

Background: N-chlorotaurine (NCT), an antiseptic that originates from the human defense system, has broad-spectrum microbicidal activity and is well tolerated by human tissue and applicable to sensitive body regions. Bacteria in short-term biofilms, too, have been shown to be killed by NCT. It was the aim of the present study to demonstrate the activity of NCT against bacteria and yeasts in longer-lasting biofilms, including their co-culture. Materials and methods: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella variicola biofilms were grown for 14 weeks in MBECTM inoculator with 96 well base. Some pegs were pinched off weekly and incubated in 1% NCT in PBS (PBS only for controls) at pH 7.1 and 37 °C, for 30 and 60 min. Subsequently, bacteria were resuspended by ultrasonication and subjected to quantitative cultures. Similar tests were conducted with C. albicans biofilms grown on metal (A2-steel) discs for 4 weeks. Mixed co-cultures of C. albicans plus each of the three bacterial strains on metal discs were grown for 5–7 weeks and weekly evaluated, as mentioned above. Results: Single biofilms of S. aureus, P. aeruginosa, and K. variicola grew to approximately 1 × 106 colony forming units (CFU)/mL and C. albicans to 1 × 105 CFU/mL. In combined biofilms, the CFU count was about 1 log10 lower. Viable counts of biofilms of single bacteria were reduced by 2.8 to 5.6 log10 in 1% NCT after 60 min (0.9 to 4.7 log10 after 30 min) with Gram-negative bacteria being more susceptible than S. aureus. Significant reduction of C. albicans by 2.0 to 2.9 log10 occurred after 4 h incubation. In combined biofilms, viable counts of C. albicans were reduced by 1.1 to 2.4 log10 after 4 h, while they reached the detection limit after 1 to 2 h with bacteria (2.0 to > 3.5 log10 reduction). Remarkably, older biofilms demonstrated no increase in resistance but constant susceptibility to NCT. This was valid for all tested pathogens. In electron microscopy, morphological differences between NCT-treated and non-treated biofilms could be found. Conclusions: NCT is active against long-term biofilms of up to several months irrespective of their age. Combined biofilm cultures of yeasts and bacteria show a similar susceptibility pattern to NCT as single ones. These results contribute to the explanation of the clinical efficacy of NCT, for instance, in infected chronic wounds and purulently coated crural ulcerations.

Pharmacokinetics and pharmacodynamics of enrofloxacin treatment of Escherichia coli in a murine thigh infection modeling

Background Enrofloxacin is an antibacterial drug with broad-spectrum activity that is widely indicated for veterinary use. We aim to develop the clinical applications of Enrofloxacin against colibacillosis by using the neutropenic mice thigh infection model. Results The minimum inhibitory concentration (MIC) distribution of 67 isolated E. coli strains to ENR was calculated using CLSI guidelines. Whereas, the MIC50 value calculation was considered as the population PD parameter for ENR against E. coli strains. The MIC values of 15 E. coli strains were found to be nearest to the MIC50 i.e., 0.25 μg/mL. Of all the tested strains, the PK-PD and E. coli disease model was established via selected E. coli strain i.e., Heilong 15. We analyzed the PK characteristics of ENR and its metabolite ciprofloxacin (CIP) following a single subcutaneous (s.c.) injection of ENR (1.25, 2.5, 5, 10 mg/kg). The concentration-time profiling of ENR within the plasma specimens was determined by considering the non-compartmental analysis (NCA). The basic PK parameters of ENR for the peak drug concentration (Cmax) and the area under the concentration-time curve (AUC) values were found to be in the range of 0.27–1.97 μg/mL and 0.62–3.14 μg.h/mL, respectively. Multiple s.c. injection over 24 h (1.25, 2.5, 5, 10 mg/kg at various time points i.e., 6, 8, 12, and 24 h respectively) were administered to assess the targeted PD values. The Akaike Information Criterion (AIC) was used to choose PD models, and the model with the lowest AIC was chosen. The inhibitory Emax model was employed to calculate the related PK-PD parameters. The results of our study indicated that there was a strong correlation between the AUC/MIC and various antibacterial activities (R2 = 0.9928). The target values of dividing AUC/MIC by 24 h for bacteriostatic action were 1-log10 reduction, 2-log10 reduction, and 3-log10 reduction 0.325, 0.4375, 0.63, and 0.95 accordingly. Conclusion The identified pharmacodynamics targets for various antibacterial effects will be crucial in enhancing ENR clinical applications and serving as a key step in reducing bacterial resistance.

Application of EN 16615 (4-Field Test) for the Evaluation of the Antimicrobial Activity of the Selected Commercial and Self-Made Disinfectant Wipes

The purpose of disinfectants is to reduce microorganisms on a contaminated surface and to prevent the spread of microorganisms. The relatively new EN 16615 simulates disinfection by wiping and allows for assessing the recovery of microorganisms from the surface and, importantly, the degree of spread of microorganisms when the surface is disinfected by wiping. For the first time, using this standard, the tested products in the form of commercial disinfectant wipes were compared with self-made wipes soaked in respective disinfectant liquids. The disinfected surfaces were simulated by homogeneous polyvinyl chloride plates. The studies were carried out not only with the standard, but also with clinical multidrug-resistant microbial strains. Based on the research, it can be concluded that the most effective products in the disinfection process (log10 reduction of ≥5) with the shortest contact time (1 min) were products containing ethanol, propanol, and quaternary ammonium compounds (self-made wipes) and propanol (commercial wipes). The least effective products (log10 reduction of <5) in terms of the contact time declared by the manufacturer were products containing ethanol and sodium hypochlorite (commercial wipes). Much better antimicrobial activity of self-made wipes was observed in comparison to the activity of the commercial wipes.

Inhibition of LpxC Increases the Activity of Iron Chelators and Gallium Nitrate in Multidrug-Resistant Acinetobacter baumannii

Infections caused by multidrug-resistant Acinetobacter baumannii would benefit from the development of novel treatment approaches. Compounds that interfere with bacterial iron metabolism, such as iron chelators and gallium nitrate, have previously been shown to have antimicrobial activity against A. baumannii. In this study, we characterize the effect of LpxC inhibitors on the antimicrobial activity of previously characterized iron chelators, 2,2′-bipyridyl (BIP) and deferiprone (DFP), and gallium nitrate (Ga(NO3)3) against A. baumannii reference strains and multidrug-resistant clinical isolates. The LpxC inhibitor LpxC-2 was synergistic with BIP for 30% of strains tested (FICI values: 0.38–1.02), whereas inhibition with LpxC-4 was synergistic with BIP for 60% of strains tested (FICI values: 0.09–0.75). In time–kill assays, combinations of BIP with both LpxC inhibitors demonstrated synergistic activity, with a more than 3 log10 reduction in bacterial counts compared to BIP alone. LpxC-2 was synergistic with Ga(NO3)3 for 50% of strains tested (FICI values: 0.27–1.0), whereas LpxC-4 was synergistic with Ga(NO3)3 for all strains tested (FICI values: 0.08–≤0.50). In time–kill assays, combinations of Ga(NO3)3 with LpxC-2 and LpxC-4 decreased the growth of both strains compared to each compound separately; however, only the combination with LpxC-4 met the defined criteria for synergy. These results identify a novel synergy between two antimicrobial classes against A. baumannii strains.