Presentation Title

A Look into Complement Sensitivity Among Gram-Positive Bacteria

Faculty Mentor

Ben Aronson

Start Date

17-11-2018 9:45 AM

End Date

17-11-2018 10:00 AM

Location

C164

Session

Oral 2

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Gram-positive bacteria were previously thought to be completely resistant to the complement proteins of the innate immune system. The main focus of these experiments is to study complement sensitivity among Gram-positive bacteria. Three strains of Gram-positive bacteria were tested for complement sensitivity: Bacillus subtilis, Bacillus megaterium, and Bacillus cereus. These model organisms are used as close comparisons to related pathogenic Gram-positive bacteria such as Staphylococcus aureus. These three strains were each grown in the presence of prepared serum treatments, and colony survival was quantitatively measured. Complement sensitivity was determined to be variable among these strains of bacteria. While two of the species were either insensitive or only slightly sensitive to complement, 30-50% of B. cereus was killed by complement, thus challenging the previous evaluation of universal complement resistance among Gram-positive bacteria (3).

1. Brown, Lisa, et al. 2015. “Through the Wall: Extracellular Vesicles in Gram-Positive Bacteria, Mycobacteria and Fungi.” Nature Reviews Microbiology, vol. 13, no. 10, pp. 620–630., doi:10.1038/nrmicro3480.

2. Chipman, D. M., and N. Sharon. 1969. “Mechanism of Lysozyme Action.” Science, vol. 165, no. 3892, pp. 454–465., doi:10.1126/science.165.3892.454.

3. Jeanneau, C., et al. 2015. “Can Pulp Fibroblasts Kill Cariogenic Bacteria? Role of Complement Activation.” Journal of Dental Research, vol. 94, no. 12, pp. 1765–1772., doi:10.1177/0022034515611074.

4. Reynolds, B. L., and D. Rowley. 1969. “Sensitization of Complement Resistant Bacterial Strains.” Nature, vol. 221, no. 5187, pp. 1259–1261., doi:10.1038/2211259a0.

Summary of research results to be presented

  1. Cultures of each bacteria were grown in Tryptic Soy Broth (TSB) in a shaking water bath at 37 C until mid-log phase.

  2. Each culture was then diluted to a concentration of 8 cells/microliter.

  3. 50 microliters of the dilution was mixed with 25 microliters of treatment: Serum [+lysozyme, +complement] ,TSB [-lysozyme, -complement], Heat-treated serum [+lysozyme, -complement], Bentonite-treated serum [-lysozyme, +complement], and Bentonite-heat-treated serum [-lysozyme, -complement].

  4. The 75 microliter solutions were incubated for sixty minutes at 37 C before being plated on Tryptic Soy Agar (TSA).

  5. Plates were grown overnight before colony count was determined.

  6. With TSB as 100% survival, each treatment group’s survival was determined, as well as complement sensitivity. Complement sensitivity should be revealed when comparing S to HT or BT to BHT.

  7. The M-W U Test was performed to determine if the difference in any pair of treatments was statistically significant.

B. subtilis was slightly susceptible to complement; B. megaterium was not susceptible; B. cereus was modestly susceptible to complement.

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Nov 17th, 9:45 AM Nov 17th, 10:00 AM

A Look into Complement Sensitivity Among Gram-Positive Bacteria

C164

Gram-positive bacteria were previously thought to be completely resistant to the complement proteins of the innate immune system. The main focus of these experiments is to study complement sensitivity among Gram-positive bacteria. Three strains of Gram-positive bacteria were tested for complement sensitivity: Bacillus subtilis, Bacillus megaterium, and Bacillus cereus. These model organisms are used as close comparisons to related pathogenic Gram-positive bacteria such as Staphylococcus aureus. These three strains were each grown in the presence of prepared serum treatments, and colony survival was quantitatively measured. Complement sensitivity was determined to be variable among these strains of bacteria. While two of the species were either insensitive or only slightly sensitive to complement, 30-50% of B. cereus was killed by complement, thus challenging the previous evaluation of universal complement resistance among Gram-positive bacteria (3).

1. Brown, Lisa, et al. 2015. “Through the Wall: Extracellular Vesicles in Gram-Positive Bacteria, Mycobacteria and Fungi.” Nature Reviews Microbiology, vol. 13, no. 10, pp. 620–630., doi:10.1038/nrmicro3480.

2. Chipman, D. M., and N. Sharon. 1969. “Mechanism of Lysozyme Action.” Science, vol. 165, no. 3892, pp. 454–465., doi:10.1126/science.165.3892.454.

3. Jeanneau, C., et al. 2015. “Can Pulp Fibroblasts Kill Cariogenic Bacteria? Role of Complement Activation.” Journal of Dental Research, vol. 94, no. 12, pp. 1765–1772., doi:10.1177/0022034515611074.

4. Reynolds, B. L., and D. Rowley. 1969. “Sensitization of Complement Resistant Bacterial Strains.” Nature, vol. 221, no. 5187, pp. 1259–1261., doi:10.1038/2211259a0.