1072. In Vitro Antibacterial Susceptibility Testing of Sulopenem Against Category A and B Bio-threat Bacterial Pathogens

Background Sulopenem is a thiopenem β-lactam antibiotic being developed for the treatment of infections caused by multi-drug resistant bacteria. Sulopenem possesses potent activity against species of the Enterobacterales that encode ESBLs or AmpC-type β-lactamases that confer resistance to third generation cephalosporins. It has also demonstrated good in vitro microbiological activity against a range of bacterial pathogens including penicillin resistant S. pneumoniae, β-lactamase-producing H. influenzae and M. catarrhalis. Sulopenem is available as intravenous and oral pro-drug formulations, and its activity aligns with the most urgent drug-resistant antimicrobial threats defined by the CDC. Methods Bacterial inoculums were prepared by suspending colonies into cation adjusted Mueller Hinton broth (CAMHB) from 18-24 h (B. anthracis, B. pseudomallei and B. mallei plates incubated at 35ºC); or 36-48 h (F. tularensis and Y. pestis plates incubated at 35ºC and 28ºC, respectively). Sheep blood agar plates were used for B. anthracis and Y. pestis. Chocolate agar plates were used for F. tularensis, B. pseudomallei and B. mallei. Suspended cultures were diluted with CAMHB to achieve a turbidity equivalent to a 0.5 McFarland standard. MICs were determined by the microdilution method in 96-well microplates according to CLSI guidelines (Clinical and Laboratory Standards Institute, 2020). Antibiotic ranges used for sulopenem were 0.03 - 64 μg/mL and 0.004 - 8 μg/mL for the diversity strains of B. anthracis, F. tularensis, Y. pesis, B. mallei, and B. pseudomallei, based on a final well volume of 100 μl after inoculation. Results A summary of sulopenem MIC90 results versus bio-threat bacterial pathogens in presented in the table. Criteria for down selection into mice was met for all pathogens except F. tularensis. Sulopenem MIC90 Summary for Down Selection Criteria Conclusion Sulopenem is active in vitro against a number of bio-threat pathogens at concentrations likely to be achieved after oral dosing in humans and meets criteria to be tested in the murine model of B. anthracis, Y. pestis, B. mallei, and B. pseudomallei. Disclosures Michael Dunne, MD, Iterum Therapeutics (Board Member, Consultant, Shareholder) Steven I. Aronin, MD, Iterum Therapeutics (Employee, Shareholder)

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The cultivated diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function compared to the allotetraploid cotton species through genetic transformation, the reliable and efficient method for high frequency somatic embryogenesis and plant regeneration in G. arboreum is urgent need to be established. Callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB (MS salts and B5 vitamins) medium with 0.09 µM 2,4-D and 2.32 µM KT. The embryogenic callus was induced on MS5 medium from the suspended cultures of several cycles of alternate liquid-solid culture, which was critical step for somatic embryogenesis. The liquid medium of MS4 was supplemented with 0.1g/L NaCl, 0.1g/L KCl and 0.1g/L CuSO4. The solid medium of MS5 for embryogenic callus effective induction was supplemented with 37.59 mM KNO3 + 62.47 µM NH4NO3 and 2.46 µM IBA + 0.93 µM KT or 0.045 µM 2,4-D + 2.46 µM IBA + 0.465 µM KT. During callus growing on different media, callus was effectively selected for subculture or treatment according to cell morphology to induce embryogenic callus and somatic embryos. Somatic embryo maturation and germination were better on MS5 medium with maltose or glucose + maltose than the single glucose. The regenerated plantlets with well-developed roots were directly transferred to soil or grafted onto the germinated cotton plantlets. The feasible process of plant regeneration via somatic embryogenesis in diploid cultivated species was established and needed to be improved and optimized for the gene functional analysis and gene editing in the diploid cotton species.

The Architecture of Monospecific Microalgae Biofilms

Microalgae biofilms have been proposed as an alternative to suspended cultures in commercial and biotechnological fields. However, little is known about their architecture that may strongly impact biofilm behavior, bioprocess stability, and productivity. In order to unravel the architecture of microalgae biofilms, four species of commercial interest were cultivated in microplates and characterized using a combination of confocal laser scanning microscopy and FTIR spectroscopy. In all the species, the biofilm biovolume and thickness increased over time and reached a plateau after seven days; however, the final biomass reached was very different. The roughness decreased during maturation, reflecting cell division and voids filling. The extracellular polymeric substances content of the matrix remained constant in some species, and increased over time in some others. Vertical profiles showed that young biofilms presented a maximum cell density at 20 μm above the substratum co-localized with matrix components. In mature biofilms, the maximum density of cells moved at a greater distance from the substratum (30–40 μm), whereas the maximum coverage of matrix components remained in a deeper layer. Carbohydrates and lipids were the main macromolecules changing during biofilm maturation. Our results revealed that the architecture of microalgae biofilms is species-specific. However, time similarly affects the structural and biochemical parameters.

Planktonic versus Biofilm Catabolic Communities: Importance of the Biofilm for Species Selection and Pesticide Degradation

ABSTRACTChloropropham-degrading cultures were obtained from sludge and soil samples by using two different enrichment techniques: (i) planktonic enrichments in shaken liquid medium and (ii) biofilm enrichments on two types of solid matrixes (plastic chips and gravel). Denaturing gradient gel electrophoresis fingerprinting showed that planktonic and biofilm cultures had a different community composition depending on the presence and type of added solid matrix during enrichment. This was reflected in the unique chloropropham-degrading species that could be isolated from the different cultures. Planktonic and biofilm cultures also differed in chloropropham-degrading activity. With biofilm cultures, slower chloropropham removal was observed, but with less build-up of the toxic intermediate 3-chloroaniline. Disruption of the biofilm architecture resulted in degradation characteristics shifting toward those of the free suspensions, indicating the importance of a well-established biofilm structure for good performance. These results show that biofilm-mediated enrichment techniques can be used to select for pollutant-degrading microorganisms that like to proliferate in a biofilm and that cannot be isolated using conventional shaken-liquid procedures. Furthermore, the influence of the biofilm architecture on the pesticide degradation characteristics suggests that for bioaugmentation the use of biofilm catabolic communities might be a proficient alternative to using planktonic freely suspended cultures.

Aerobic TCE degradation by encapsulated toluene-oxidizing bacteria, Pseudomonas putida and Bacillus spp.

The degradation rates of toluene and trichloroethylene (TCE) by Pseudomonas putida and Bacillus spp. that were encapsulated in polyethylene glycol (PEG) polymers were evaluated in comparison with the results of exposure to suspended cultures. PEG monomers were polymerized together with TCE-degrading microorganisms, such that the cells were encapsulated in and protected by the matrices of the PEG polymers. TCE concentrations were varied from 0.1 to 1.5 mg/L. In the suspended cultures of P. putida, the TCE removal rate decreased as the initial TCE concentration increased, revealing TCE toxicity or a limitation of reducing power, or both. When the cells were encapsulated, an initial lag period of about 10–20 h was observed for toluene degradation. Once acclimated, the encapsulated P. putida cultures were more tolerant to TCE at an experimental range of 0.6–1.0 mg/L and gave higher transfer efficiencies (mass TCE transformed/mass toluene utilized). When the TCE concentration was low (e.g., 0.1 mg/L) the removal of TCE per unit mass of cells (specific removal) was significantly lower, probably due to a diffusion limitation into the PEG pellet. Encapsulated Bacillus spp. were able to degrade TCE cometabolically. The encapsulated Bacillus spp. gave significantly higher values than did P. putida in the specific removal and the transfer efficiency, particularly at relatively high TCE concentration of approximately 1.0±0.5 mg/L. The transfer efficiency by encapsulated Bacillus spp. in this study was 0.27 mgTCE/mgToluene, which was one to two orders of magnitude greater than the reported values.

Eradication of Bacteria in Suspension and Biofilms Using Methylene Blue-Loaded Dynamic Nanoplatforms

ABSTRACT The bacterial killing efficiency of a dynamic nanoplatform (DNP) was evaluated. The polyacrylamide (PAA) hydrogel matrix of the DNP was loaded with methylene blue (MB) and was previously applied successfully to killing rat C6 glioma tumor cells in culture. This series of experiments is aimed at determining the suitability of this nanoplatform for elimination of bacterial infections. Suspended cultures of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter sp. were exposed to activated (∼650-nm laser light) MB-PAA-DNPs. The killing efficiency of nanoparticle mass concentration, light irradiance and fluence, and dark incubation time was determined on each of the bacterial species. Moreover, the ability of activated MB-PAA-DNPs to inhibit biofilm growth and eradicate and disperse preformed biofilms, preformed on glass and polystyrene surfaces, was demonstrated. The data revealed that activated MB-PAA-DNPs eradicated all species of bacteria examined. Also, encapsulation of MB into the PAA-DNP matrix significantly diminished the observed dark toxicity of free dye. The photobactericidal efficacy of MB-PAA-DNP was found to be higher for gram-positive bacteria than for gram-negative bacteria. In addition, activated MB-PAA-DNP can inhibit biofilm growth and eradicate almost all of the early-age biofilms that are formed by all of the bacteria examined.

Preparation and Characterization of Biodegradable β-TCP-Based Composite Microspheres as Bone Tissue Engineering Scaffolds

Since a small globular particle was first used as support for three-dimensional (3D) growth of anchorage-dependent cells in suspended cultures, a variety of microspheres as tissue engineering scaffolds have been developed. In this paper, β-TCP and chitosan were selected as the components of microspheres due to their biodegradability and osteogenic properties. The biodegradable β-TCP/chitosan composite microspheres were prepared by a solid-in-water-in-oil (s/w/o) emulsion cross-linking method in this paper. The size distribution, surface morphology, and microstructure of the microspheres were evaluated. Scanning electron microscopy revealed that the size of the microspheres with good spherical morphology was distributed in the range of 50~200μm. In vitro immersion experiments were carried out to evaluate the degradability of the microspheres, and the results demonstrated that the chitosan/β-TCP composite microspheres were potential materials as tissue engineering scaffolds for bone repair.