Invasive capability of Streptococcus uberis field isolated with different Biofilm forming profiles

MOLIVA MV; CAMPRA N; MONTIRONI ID; REINOSO EB

San Luis

Congreso; Congreso Argentino de Microbiología General SAMIGE; 2018

Bovine mastitis is a cattle disease that causes large annual economic losses around the world. Different pathogens, classified as environmental and contagious, are associated with the disease as their ability to form biofilm leads to the advance of the infection. Streptococcus uberis is one of the most important environmental pathogen. The aim of this study was to evaluate the ability to adhere and internalize of eight S. uberis field isolates with different biofilm forming capacity. Invasion assays were performed using a bovine mammary epithelial (MAC-T) cell line. Previously, we characterized the S. uberis according to the biofilm formation ability. We selected S. uberis strains according to the biofilm profile: one weak strain, three moderate strains and four strong strains. Adherence and internalization ability of the strains was determined with 5.0x10 4 MAC-T cells/well cultured in 96-well plates for 24 h. The cells were washed and co-cultured with 200 µl of the bacterial suspensions (approximately 1 x 10 6 CFU/ml) during 2 h. Then, cells were lysed with Triton 0,025% and MAC-T lysates were 10-fold serially diluted and plated on Tripticase Soya Agar. A parallel assay was performed to evaluate the internalization. After 2 h of co-culture with bacterial suspensions, cells were incubated for 1 h in DMEM culture medium supplemented with gentamicin (100 µg/ml) to eliminate adhered extracellular bacteria. Then, cells were washed and lysed. An aliquot of the lysates was collected and serial 10 fold-dilutions were made to determine the internalized CFUs per well. Our results showed that all the S. uberis assayed were able to adhere MAC-T cells. No statistical differences were found among the strains suggesting that the ability to adhere depended on the strain. In addition, all S. uberis strains were able to internalize MAC-T cells, although no differences in invasion capacity among strains were observed. Furthermore, the ability to internalize was not associated with the biofilm profile. The results provide a better understanding of virulence factors of this important environmental pathogen associated with mastitis, and may contribute to help the design of new therapeutic approaches.