Olivia S Harlow1, Ethan Houskamp1, Natalie Anumolu1, Shannon Manning, PhD2, Erica Boldenow, PhD1

10848-1calvingraphics@gmail.com

• Group B Streptococcus is a leading infectious cause of neonatal morbidity and mortality in the US1

• Ascending infection in the mother can cause adverse birth outcomes2,3

• GBS is defined as being beta-hemolytic, but about three percent of strains are non-hemolytic4

• Recent data suggests that non-hemolytic strains can also pose health risks to infants5

• Previously, we have seen that non-hemolytic strain GB37 displays hemolytic activity in liquid suspension

• We hypothesize that non-hemolytic strains cause inflammation, oxidative stress, and invasion/adhesion in cells similarly to hemolytic strains

Non-hemolytic and hemolytic Group B Streptococcus: interactions with cells in vitro1Department of Biology, Calvin University, Grand Rapids, Michigan, 2Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI

Background

https://www.rocking-mama.com/2018/01/17/baby-basics/

Methods

Results

Conclusions Acknowledgments and References

We thank Lori Keen for her outstanding managerial support, and Dr. Shannon Manning for our GBS strains. We also thank Calvin University and the Wierdafamily for supporting this research.1. Verani, J. R., et al. (2010). "Prevention of perinatal group B streptococcal disease--revised guidelines from CDC, 2010."

MMWR Recomm Rep 59(RR-10): 1-36. 2. Lawn, J. E., et al. (2017). "Group B Streptococcal Disease Worldwide for Pregnant Women, Stillbirths, and Children:

Why, What, and How to Undertake Estimates?" Clin Infect Dis 65(suppl_2): S89-S99. 3. Deutscher, M., et al. (2011). "Incidence and severity of invasive Streptococcus pneumoniae, group A Streptococcus, and

group B Streptococcus infections among pregnant and postpartum women." Clin Infect Dis 53(2): 114-123. 4. Rodriguez-Granger, J, Spellerberg, B, Asam, D, Rosa-Fraile, M. (2015) “Non-haemolytic and non-pigmented Group B

Streptococcus, an infrequent cause of early onset neonatal sepsis.” Pathog Dis 73(9): ftv089. 5. Gendrin, C., et. al. (2018) “A Nonhemolytic Group B Streptococcus Strain Exhibits Hypervirulence.” J of Infect Dis

217(1):983-987. 6. Korir, M. L, et al. (2014). “Association and Virulence Gene Expression Vary among Serotype III Group B Streptococcus

Isolates following Exposure to Decidual and Lung Epithelial Cells” Infect Immun. 82(11):4587-4595.

Non-hemolytic and Hemolytic GBS

qPCR6

Cells (Macrophages, Trophoblast, Lung Epithelial)

2-4 h co-culture (MOI of 5)

Hemolysis assay

add to add to

Red Blood Cells

DCF assay,ELISA (IL-1β),

LDH assay,Invasion/adhesion assay6

• GB37 relies on cylE upregulation to cause hemolysis of RBCs in liquid culture

• IL-1β is released from macrophages independent of hemolytic activity

• Invasion/adhesion less prominent in non-hemolytic strains, but neither strain type causes cell death

• Because no significant ROS seems to be produced in response to GBS, ROS is likely not required as a mechanism of GBS-prompted inflammation

GB 3 7

GB 2 7 9

GB 1 4 5 5

A 9 0 90

1 0

2 0

3 0

4 0

cy

lE E

xp

res

sio

n

N on -h em o ly tic

H em o ly tic

C o n trol

GB 3 7

GB 2 7 9

GB 1 4 5 5

A 9 0 9

GB 1 1 2

GB 4 1 1

GB 5 9 0

0

5 0

1 0 0

1 5 0

He

mo

lys

is (

%)

N on -h em o ly tic

H em o ly tic

Figure 2. GBS hemolytic activity correlates to differential cylE expression. A. GB37 upregulates cylE in liquid culture. (Mean +/- SEM, N=3 for GB37, N=2 for remainder) B. GB37 has hemolytic activity in liquid culture. (Mean +/- SEM, N=2)

A.

B.

GB 37

GB 27 9

GB 14 55

A 90 9

GB 11 2

GB 41 1

GB 59 0

0

100000

200000

300000

400000

500000

CFU

In vas ion

A d he s io n

N on -h em o ly tic H em o ly tic

Figure 3. GBS causes IL-1B release in macrophages (THP-1) independent of hemolytic activity. (Mean +/-SEM, N=5, *p<0.05, Friedman’s test)

Figure 5. Hemolytic GBS invades and adheres to cells better than non-hemolytic GBS. A. GB411 shows more adhesion to trophoblast cells (HTR-8), whereas GB37 invades greater than other strains. (Mean +/- SEM, N=5, two-way ANOVA) B. Non-hemolytic GBS do not adhere to or invade lung epithelial cells (A549) as well as hemolytic GBS. (Mean +/- SEM, N=1)

Figure 6. Neither trophoblast cells (A, HTR-8) nor lung epithelial cells (B, A549) exhibit cytotoxicity when co-cultured with either hemolytic or non-hemolytic GBS over a period of 4 hours. (Mean +/- SEM, N=1)

Figure 1. A909, GB112, GB411 & GB590 show characteristic hemolytic clearance and pigmentation whereas GB37 shows none. Figure 4. GBS does not cause ROS in either trophoblast

cells (HTR-8) or macrophages (THP-1, data not shown). (Mean +/- SEM, N=2)

30 minute co-culture(1x109 CFU/mL)

GB 37

GB 27 9

GB 14 55

GB 41 1

0

500

1000

15005000

10000

15000

CFU

In vas ion

A d he s io n

N on -h em o ly tic H em o ly tic

A.

B.

B.A.

Med ia

GB 3 7

GB 2 7 9

GB 1 4 5 5

A 9 0 9

GB 1 1 2

GB 4 1 1

GB 5 9 0

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

IL-1b

(p

g/m

L)

N on -h em o ly tic

H em o ly tic

*

*

*