Bioactivity of purified antibacterials secreted by entomopathogenic bacteria

1. Bioactivity of purified antibacterials secreted by entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus luminescens Matt Bowen, Floyd Inman III and Leonard Holmes Sartorius-stedim Biotechnology Laboratory, Biotechnology Research and Training Center University of North Carolina at Pembroke, Pembroke, North Carolina 28372 Purification Background Effects of Pigment Production Results and Discussion For the minimum inhibitory dilutions (MID), sterile paper disk were impregnated with decreasing dilutions of antibacterial products produced by each species of bacteria. For P. luminescens the pigmented culture had a greater bioactivity against B. megaterium than the non-pigmented culture. The MID for the pigmented culture was determined to be 20 mg/mL while the MID for the non-pigmented culture was determined to be 40 mg/mL. The antibacterial products secreted by X. nematophila were more effective against B. megaterium, than the products secreted from P. luminescens. As seen in Figure 6 the inhibition halos surrounding the disk began to appear around 10 mg/mL. Past 40 mg/mL the dilution did not make any difference in the size of the halo. By isolating the antibacterial products, and using purification techniques, we can determine minimum inhibitory concentrations of the overall antibacterials produced by these bacteria towards B. megaterium. These products are crucial for protecting the insect cadaver from an outside bacterial infection. Additionally, these compounds may be exploited for human and animal use. Antibacterial products from both bacterial species are known to be broad spectrum which means that they can be used for either Gram-positive or Gram-negative bacteria. Exploring new sources of antimicrobials is essential due to the ever increasing antimicrobial resistance of many bacterial pathogens (i.e. Staphylococcus aureus, Bacillus spp., Pseudomonas aeruginosa, etc). Wild-type P. luminescens produces a bioactive red pigment; however, and non-pigment producing mutant was isolated to observe if the red pigment produced increases bioactivity of other secreted antimicrobials. The presence of the red pigment is shown (Figure 5) to increase bioactivity. Cell cultures of both species were subjected to tangential flow filtration. A Sartorius-stedim Sartojet was used to remove cells from the liquid culture. Culture permeates were pumped through a column filled with Amberlite XAD 1180 absorbent, eluted with ethanol and allowed to dry. Both bound and unbound samples were assayed for activity (Figure 3). Xenorhabdus nematophila and Photorhabdus luminescens (Figure 1)are entomopathogenic, Gram-negative enteric bacteria, that are symbiotically associated with insect parasitic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora, respectively. Both bacteria utilizes its’ nematode symbiont to gain access to the host insect hemolymph. During proliferation, these bacteria secrete an array of toxins and enzymes that kill and bioconvert the insect for bacterial and nematode nutrition, as well as antibacterial products. These antibacterial products are produced to ensure the survival of the bacteria and their nematode partners within an insect cadaver. This study tested the bioactivity of the crudely purified antibacterial compounds secreted from 24 hour cultures of both bacteria. These products have shown to be effective against both gram positive and gram negative bacteria and are thereby described as having broad spectrum activity. Antibacterial products were isolated by subjecting cultures to tangential flow filtration, then passing culture permeates through an absorbent resin column, eluted and dried. A B Figure 5: A) Non-pigmented, mutant culture of P. luminescens with antibacterial products. B) Pigmented culture with antibacterial products. Zones are increased with red pigment indicating that red pigments enhances bioactivity Minimal Inhibitory Dilutions Figure 3:Mueller Hinton plate with a culture of B. megaterium assaying compounds isolated from X. nematophila. Disks are read clockwise: Negative control (upper left); Culture permeate (upper right); Bound compounds (lower right); Unbound compounds (lower left). Minimum inhibitory dilutions for antimicrobials of both entomopathogens (Figure 6 a, b) were determined using a modified Kirby-Bauer disk diffusion method. Dilutions of a 60 mg/mL concentrated stock of crudely purified antibacterials were tested against B.megaterium. Figure 1: Gram stain of Photorhabdus luminescens (Gram-negative bacilli) Bioactivity Assays Acknowledgements Antibacterial products from X. nematophila and P. luminescens were tested against Bacillus megaterium, a Gram-positive soil dwelling bacterium. A modified Kirby-Bauer disk diffusion method was used to test for bioactivity (zones of sensitivity) of the bound and unbound samples. Zones of inhibition were observed and measured for all samples purified from both species, and compared for overall activity against B. megaterium. Partial financial support was provided in part by the: North Carolina Biotechnology Center (grant # 2010-IDG-1008), UNC-Pembroke Department of Chemistry & Physics and Farm Bureau of Robeson County. Additionally, thanks is given to the UNCP Office of the Provost and Academic Affairs and to the following sponsors. Materials and Equipment Figure 7: Gram stain microphotograph of Bacillus anthracis, the causative agent of anthrax. During the Amerithrax incident in 2001, endospores (white structures within cells) contained in sealed envelopes were released into the US mail system, 11 people died. Development of prophylactic countermeasures, representative of this research, are necessary to thwart similar biological attacks and natural disease. Symbiotic bacteria were indirectly isolated from its nematode partner utilizing an insect host. X. nematophila and P. luminescens were up-scaled accordingly to obtain an inoculum for bioreactor production (Figure 2). Figure 6: Antimicrobial products in dilutions of; 5, 10, 20, 30, 40, 50 and 60 mg/mL (clockwise). Negative control (center): A) P. luminescens. B) X. nematophila Figure 4:P. luminescens bioactivity assay against B. megaterium (read clockwise). Top, Negative control disk; Right, culture permeate, Bottom, absorbed antimicrobials, Left, resin flowthrough. Figure 2: Culture of P.luminescens grown in a 2L Sartorius bioreactor.