Materials and Methods

1
Optimization of Production of Antimicrobial
Agents by Actinomycete Strain NC-18 Against
Methicillin-Resistant Staphylococcus aureus
(MRSA) Adam Klassen Supervisor Dr. Naowarat
Cheeptham Co-supervisors Dr. Kingsley Donkor
and Joanna Urban

• Introduction
• MRSA is responsible for 20-30 of nosocomial
  infections1
• MRSA is resistant to most available antibiotic
  treatments2
• NC-18, an actinomycete strain, has shown activity
  against MRSA3
• Optimization of fermentation conditions is
  required for high throughput production of
  antimicrobial agents
• The objective of this project was to determine
  the optimum media and parameters for NC-18
  fermentation that will maximize production of
  antimicrobial agents

Figure 1 Disk diffusion results for the 5 media.
Paper disks were impregnated with 60µL of the
medium sample and left to dry. Dried disks were
placed on 0.5 McFarland standard MRSA plates and
incubated at 35ºC for 24 hours. An asterix beside
the medium in the legend denotes incomplete zones
of inhibition, while no asterix denotes complete
inhibition.
Figure 3 Media color after 9 days of incubation
with NC-18. Left to right YMG, YpSs, GOT, MH,
FR-23.

• Materials and Methods
• Media Optimization
• YMG, YpSs, GOT, MH, and FR-23 media were used for
  NC-18 fermentation
• 100 mL of each medium was adjusted to pH 7 before
  inoculation with NC-18 and incubated in a shaker
  set at 250 rpm at 30ºC for 9 days
• Five samples of 5 mL were taken for MRSA disk
  diffusion assay and to monitor pH and percent
  packed cell volume (PCV)
• MRSA disk diffusion assay was utilized to
  determine the optimum medium for production of
  antimicrobial agents based on zones of inhibition
• Parameter Optimization (Response Surface
  Methods4)
• Media volume, pH, and carbon source levels of the
  optimum medium were studied
• 15 flasks with varying volume, pH, and amount of
  carbon source were inoculated with NC-18 and
  incubated in a shaker set at 250 rpm at 30 ºC for
  10 days
• Five samples of 5 volume were taken for MRSA
  disk diffusion assay and to monitor pH and PCV
• MRSA disk diffusion assay was utilized to
  determine optimal parameters for production of
  antimicrobial agents

• Discussion
• A more sensitive assay for the detection of the
  antimicrobial agents of interest is needed
• Other parameters such as nitrogen source,
  phosphate source, temperature, and shaking speed
  need to be investigated in order to fully
  optimize YMG

• Future Research
• Experimentally implement an assay for
  antimicrobial agents by coupling disk diffusion
  assay with CE/MS
• Possibly employ other experimental media to
  compare with YMG results
• Determine molecular characteristics of
  antimicrobial agents such as structure through
  isolation and purification

Figure 2 Disk diffusion assay results for YMG
parameter optimization. Disk diffusion assay
procedure was done as described above in Figure 1.
Acknowledgements Special thanks to my supervisors
and Carolynne Fardy for their patience and help
when nothing seemed to work. Thanks as well to
previous students for their research on NC-18 and
to Traders Protein for supplying free samples of
Pharmamedia.

• Results
• YMG medium was found to be the optimum medium for
  NC-18 production of antimicrobial agents that
  inhibit MRSA growth (Figure 1)
• Similar general trend of increasing PCV and pH
  with production of antimicrobial agents was seen
• Optimization results showed that F6 had the
  highest production of antimicrobial agents
  (Figure 2)
• Media with higher pH and lower volume had
  increased production of antimicrobial agents.
  Glucose levels had no visible effect on production

• References
• Gunnarsson et al (2003). Journal of Industrial
  Microbiology and Biotechnology 30 150-156.
• Francis et al (2005). Clinical Infectious
  Diseases 40 100-107
• Cheeptham et al (2005). The 55th Canadian Society
  of Microbiology Conference. Dalhousie
  University, Halifax. June 12 to 15.
• Box et al (1978). Statistics for experimenters.
  Wiley-Interscience. p 510