Hemolytic Activity of Bacterium

Last week, we continued to study our unknown soil microbe by performing a Hemolysis Test. The hemolysis test helped us determine whether our bacteria is said to be fastidious. Fastidious organisms require a rich growth medium filled with specific nutrients required to grow (Lab Handout). The medium used for this test is a Blood Agar, composed of general nutrients and 5% sheep blood. Bacteria that flourish on this blood agar produce exoenzymes called helolysins, which trigger the lysing of red blood cells. Blood agar tests are especially useful in determining streptococcal species. Based on their hemolytic activity response to the blood agar, bacteria can be categorized into three different groups:

Type	Meaning	Appearance
Beta (β) hemolysis	Complete or true lysis of red blood cells	Clear zone, almost transparent of the base medium, surrounds colonies (due to destruction of hemoglobin released from erythrocytes)
Alpha (α) hemolysis	Reduction of red blood cell hemoglobin to methemoglobin in medium surrounding colony	- Green or brown discoloration in medium (brusing color) - Cell membrane in tact
Gamma (γ) hemolysis	Lack of hemolysis	No reaction surrounding the medium

Below are photographs of our controls, Staphylococcus aureus and Staphylococcus epidermis, and our unknown bacterium after 48 hours. Although it is not fully clear which hemolytic reaction our unknown bacterium belongs to, it is most closely related to an Alpha (α) hemolysis reaction. The dark coloring surrounding the colonies is a good indicator of alpha hemolysis. After seven days, the plates were observed again to see if our prediction on alpha hemolysis was correct. After a prolonged incubation, many alpha hemolytic organisms begin to appear clearer, with still a surrounding bruised color (Lab Handout). Pictures of our unknown bacterium after a prolonged seven-day incubation are below. Our microbe did not lyse the red blood cell, and kept the cell membrane in tact.

 

S. aureus

S. epidermis

Unknown Bacterium - 48 hrs. 

Unknown Bacterium - Day 7

Hemolysins have the capability to lyse red blood cells through several mechanisms. One way this happens is through the formation of pores in phospholipid bilayers (Chalmeau et al, 2011). The pores in the phospholipid bilayer allow extracellular fluids or components, such as bacteria, to enter in the red blood cell and lyse. Many of the hemolysins are categorized as pore-forming toxins, which not only lyse red blood cells, but leukocytes, and platelets. Hemolysins also function as enzymes. They damage the membrane of the red blood cell by cleaving the phospholipid in the membrane (Honda et al, 1985). This could potentially alter the structure of the membrane, allowing swelling of the red blood cell and eventually lysing.

Virulence factors are anything that could potentially produce a disease in humans. When comparing hemolytic microbes to non-hemolytic microbes, hemolytic microbes serve as potential virulence factors when combined with other factors, and therefore more virulent than non-hemolytic microbes. The lysing of red blood cells may not be hazardous initially, but combined with other bacterial factors, the possibility of virulence increases (Woltjes and de Graff, 1983). Typical soil microbes may be hemolytic, breaking down red blood cells for nutrients to encourage growh of the bacteria. The breakdown of red blood cells is used when a physician tests for strep throat, when looking for the streptococci bacteria (Chapter 5).

           

According to the dichotomous key and the idea that our unknown soil microbe is alpha-hemolytic, our bacteria could be Streptococcus pneumoniae, which is different than our test from last week, where we believed our bacteria would be Actinomyces spp. With one week and one final test left, we hope to finally narrow down what our unknown bacterium is combining our research into discovering our culprit.

 Chalmeau, J., N. Monina, J. Shin, C. Viey, V. Noireaux. (2011) α-Hemolysin pore formation into a supported phospholipid bilayer using cell-free expression. Biochemica et Biophysica Acta (BBA) – Biomembranes. 1808(1), 271-278.

Honda, T., M. Yoh, U. Kongmuang, T. Miwatani. (1985) Enzyme-linked imunosorbent assays for detection of thermostable direct hemolysin of Vibrio parahaemolyticus. Journal of Clinical Microbiology. 22(3): 383-386.

Weeks, B. I. Edward. Alcamo. Microbes and Society. Sudbury, MA: Jones and Bartlett, 2008. Print.

Woltjes J., J. de Graaf. (1983) Virulence of beta-hemolytic and non-hemolytic Streptococcus mutans: lethal dose determinations in neonatal mice. Antonie Van Leeuwenhoek. 49(4-5): 353-360.