DIVERSITY OF CRISPR SYSTEMS IN STRAINS OF STREPTOCOCCUS THERMOPHILUS UTILIZED IN ARGENTINA

PEDRÓN, LARA; PUJATO, SILVINA; QUIBERONI ANDREA; MERCANTI, DIEGO

Congreso; 32 CONGRESO BRASILERO DE MICROBIOLOGÍA; 2023

The growth of lactic acid bacteria (LAB) during the production of a fermented food increases its shelf life and is favorable for the production process, its hygienic characteristics and its sensory profile. The main LAB used in the Argentine dairy industry is Streptococcus thermophilus. Like any bacteria used to make food, it is harmed by the attack of (bacterio)phages. Within the bacterial defense systems against phages, in this particular species the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems stand out, extensively studied in strains of S. thermophilus in other countries, but not in Argentina. These systems incorporate short segments (called spacers) of exogenous DNA, such as from a phage, rendering the bacterial cell and its progeny immune to future infections by said phage, or another relative that shares said DNA sequence. Using PCR using universal primers, in the present study CRISPR1 and CRISPR3 systems were searched (the two known and active in S. thermophilus), and they were found in 35 and 40 strains, respectively, of 79 analyzed; 21 of these strains presented both systems, and in 22 strains neither was detected. 13 CRISPR1 loci were sequenced, containing 77 different spacers, 48 found in reported phage genomes, and 1 in plasmids. Regarding CRISPR3, 28 sequenced loci contained 183 distinct spacers, 123 found in phage genomes, and 7 in plasmids. Of the total spacers, there were 36 new ones (not found in databases): 6 from CRISPR1, found in a single local strain, and 30 from CRISPR3, distributed in 19 strains. The comparison of spacers between our strains and those reported in other countries shows a high similarity. However, in the local strains, a significantly lower acquisition of new and different spacers was observed in the CRISPR1 systems, which would indicate low activity, or bacterial resistance to infection by the majority of phages in their environment. Unlike previous reports, we found a higher proportion of unique arrangements for CRISPR3 with respect to CRISPR1, possibly indicating greater activity of CRISPR3 systems in nationally circulating strains, although the spacer arrangements are similar to those already reported in S. thermophilus . The presence of spacers was analyzed in a pool of 66 bacteriophage genomes reported in the database; CRISPR1-sp22 and CRISPR3-sp30 were the most conserved spacers in said pool, being found in 12 and 19 phages in the pool, respectively. Our results allow us to understand the diversity of CRISPR systems in S. thermophilus strains circulating in our country, generating an important resource for the design of strategies to reduce the impact of bacteriophages in the regional dairy industry.