The Effects of Co-culturing on In Vitro Soil Bacterial Cultivability and the Discovery of Novel Antibiotics

Since the 1928 discovery of penicillin by Alexander Fleming, antibiotics have been an integral component of healthcare. However, within a few years of use, resistant bacterial strains were found against all utilized antibiotics which limited their therapeutic efficacy. Increased prevalence of infections caused by resistant species led the Centers for Disease Control and Prevention (CDC) to release an expanded list of antibioticresistant priority pathogens (AARPs) in 2013 and in 2017 the World Health Organization (WHO) reorganized this list yet again. Since environmental microbes remain an optimal source of antibiotics, new microbial culturing methods have been developed to increase microbial yield and biodiversity. However, these novel methods required protracted experimentation and lack experimental control due to use of in situ incubation. The purpose of this study was to develop a microbial co-culturing system that allowed diffusion of metabolites between environmental microbes in a laboratory setting with the goal of increasing microbial yield and biodiversity in comparison to traditional in vitro culturing methods. Soil samples from Radium Springs, Georgia, USA, a sparsely studied site, were co-cultured utilizing semi-solid agar inoculation, a novel culturing technique. Isolated soil microbes were then co-cultured with both multi-drug-resistant Acinetobacter baumannii (MDR-AB), methicillin-resistant Staphylococcus aureus (MRSA) to determine whether the environmental microbes possessed any antibiotic properties against these AARPs. Plate analysis of co-cultured environmental samples revealed that the novel technique yielded more microbes with diverse colony morphologies at a higher density than similar samples cultured using serial dilution, filter, and filtrate culturing methods. Plate analysis of co-cultured pathogen samples revealed several microbes with inhibitive properties. This technique could prove especially powerful in increasing bacterial yield from environmental samples and aid in antibiotic research and discovery for antibiotic-resistant priority pathogens (ARPPs

Utilization of Semi-Solid Agar Inoculation in Microbial Co-culture Yields a Bacteria with Antibiotic Properties Against MRSA

Microbial co-culture has many important applications, not least of which being the identification of novel antibiotic factors. Semi-solid agar inoculation co-culture is an innovative technique that aims to increase the yield and biodiversity of cultured environmental samples and to physically separate bacteria to aid in the isolation process. Methicillin-resistant Staphylococcus aureus (MRSA) inoculated into semi-solid tryptic soy agar (TSA) with a 1% agar concentration was able to proliferate and disseminate throughout the entirety of the plate without difficulty. Bacteria isolated from soil obtained from Radium Springs, Georgia was cultured onto MRSA-inoculated agar using a point inoculation technique. Isolates of interest were identified by their ability to produce a zone of inhibition whereby inoculated MRSA had a decrease or absence of growth in the region containing the proliferating soil colony and 1-2 mm beyond its borders. Due to the ability of secreted factors to diffuse throughout the semi-solid agar, interactions between the microorganisms, or lack thereof, were readily seen. Results revealed a Gram-positive bacteria that demonstrated inhibitory effects in multiple co-culture experiments. Preliminary characterization showed the inhibitory bacteria to be bacillus shaped and non-spore-forming amongst other properties. Current findings suggest that this bacterium may be the source of a novel antibiotic with the potential to inhibit MRSA

Effects of Mass Culture Technique on Microbial Yield and Biodiversity

The great plate count anomaly describes the phenomena whereby the yield and diversity of microbes cultured from environmental samples are lower than expected. The difference in expected versus obtained results is due to many factors, including lack of access to environmental nutrients and change in growth conditions. This study examined the effect of various culturing methods on both biodiversity and yield of environmental samples. Culturing techniques utilized included serial dilution plating, filter stamping, filtrate culturing, and semi-solid agar inoculation co-culture. Each technique alters the density of the microbes in the environmental sample either by changing the sample concentration or excluding by size. In addition, co-culturing increases likelihood that growth factors will be produced. Microbial density produced by each method was comparable but differed based on the degree of sample dilution. The diversity of cultured samples, defined by differences in colony morphology, was greatest for the serial dilution plating and co-culturing methods. Results indicate that size exclusion, as well as differences in sample concentration of the various culturing methods, has a great effect on microbe biodiversity in mass culture. Due to the urgent need for novel antibiotics, understanding the effect of mass culturing methods on microbial yield can aid in determining the experimental design for isolating antibiotic-producing microbes from environmental samples

Development of Amino Acid Prodrugs of Cytarabine

Cytarabine is widely used in chemotherapy for different types of leukemia due to the targeting effects on cells in the S phase of cell cycle. However, its use is limited by its poor absorption and fast degradation to biologically inactive forms, and elimination. Amino acid (isoleucine and valine) prodrugs of cytarabine were synthesized to improve uptake of cytarabine. The prodrugs may also reduce the proportion of the drug that was degraded and increase bioavailability. Stability and permeability studies performed with cytarabine prodrugs demonstrated improved uptake. Cytotoxic studies utilizing K562 cell lines revealed 4.5% greater growth inhibition at 24 hours incubation and 19% greater growth inhibition at 72 hours incubation with the cytarabine isoleucine prodrug than with cytarabine. The results indicated that the prodrug may work more efficiently than the parent drug