Revisiting typing systems for group B Streptococcus (GBS) prophages: an application in prophage detection and classification in GBS isolates from Argentina

Group B Streptococcus (GBS) causes severe infections in neonates and adults with comorbidities. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on the presence and diversity of prophages in GBS isolates from humans in South America. This study provides insights into the prophage content of 365 GBS isolates collected from clinical samples in the context of an Argentinean multicentric study. Using whole genome sequence data, we implemented two previously proposed methods for prophage typing: a PCR approach (carried out in silico ) coupled with a blastx-based method to classify prophages based on their prophage group and integrase type, respectively. We manually searched the genomes and identified 325 prophages. However, only 80% of prophages could be accurately categorised with the previous approaches. Integration of phylogenetic analysis, prophage group and integrase type allowed for all to be classified into 19 prophage types, which correlated with GBS clonal complex grouping. The revised prophage typing approach was additionally improved by using a blastn search after enriching the database with 10 new genes for prophage group classification combined with the existing integrase typing method. This modified and integrated typing system was applied to the analysis of 615 GBS genomes (365 GBS from Argentina and 250 from public databases), which revealed 29 prophage types, including 2 novel integrase subtypes. Their characterization and comparative analysis revealed major differences in the lysogeny and replication modules. Genes related to bacterial fitness, virulence or adaptation to stressful environments were detected in all prophage types. Considering prophage prevalence, distribution and their association with bacterial virulence, it is important to study their role in GBS epidemiology. In this context, we propose the use of an improved and integrated prophage typing system suitable for rapid phage detection and classification with little computational processing. Author summary Bacteriophages, which are viruses that infect bacteria, exert a profound influence on microbial evolution when integrated into the bacterial genome, a state in which they are called prophages. It has been proposed that prophage acquisition played a role in the emergence of Streptococcus agalactiae (GBS) as a human pathogen in European countries. Further study and characterization of prophages of GBS from around the world would provide valuable insights into the mechanisms underlying GBS adaptation, evolution and epidemiology. Unfortunately, existing tools for prophage screening exhibit limitations in the detection and classification of all prophages present in GBS genomes. To address this issue, in this work we propose a new prophage typing system that allows the detection and classification of GBS prophages based on both their phylogenetic lineage and integration site within the bacterial genome. Using this methodology we were able to identify 29 prophage types in 615 GBS isolated globally. We further characterised these prophages and found that they carried genes that could give an evolutionary advantage to their host and that different lineages of GBS carried different prophage types. Comprehensive exploration and characterization of prophages represent an indispensable endeavour, providing critical insights into microbial evolution, epidemiology, and potential therapeutic interventions. ### Competing Interest Statement The authors have declared no competing interest.