biostudies-literatureJuanpere-Borras MResearch Center for Genetic Engineering, National Research and Innovation Agency (BRIN), Bogor, IndonesiaChina Scholarship CouncilEuropean Union’s Horizon 2020 research and innovation program2600145https://www.ebi.ac.uk/biostudies/studies/S-EPMC12707516biostudies-literatureUnknown17(1)<i>Streptococcus suis</i> is a major cause of sepsis and meningitis in pigs, and zoonosis through the emergence of disease-associated lineages. The ability of <i>S. suis</i> to adapt and survive in host environments, such as blood and cerebrospinal fluid (CSF), is important for pathogenesis. Here, we used Transposon Sequencing (Tn-seq) coupled with Nanopore sequencing to identify conditionally essential genes (CEGs) for the growth of <i>S. suis</i> P1/7 in active porcine serum (APS) and CSF derived from choroid plexus organoids. To our knowledge, this is the first successful application of ONT to Tn-library screening, enabling rapid local runs and a publicly available analysis pipeline. Through comparative fitness analyses, we identified 33 CEGs that support growth in APS and 25 CEGs in CSVirulenceGenome-wide Identification of conditionally essential genes supporting &lt;i&gt;Streptococcus suis&lt;/i&gt; growth in serum and cerebrospinal fluid.PMC12707516201906350084956154Fernandez-Ciruelos BWells JMArifa NBoekhorst JZhao TWagenaar Tvan Baarlen PJuanpere-Borras MSanyal RfalseGenome-wide Identification of conditionally essential genes supporting &lt;i&gt;Streptococcus suis&lt;/i&gt; growth in serum and cerebrospinal fluid.<i>Streptococcus suis</i> is a major cause of sepsis and meningitis in pigs, and zoonosis through the emergence of disease-associated lineages. The ability of <i>S. suis</i> to adapt and survive in host environments, such as blood and cerebrospinal fluid (CSF), is important for pathogenesis. Here, we used Transposon Sequencing (Tn-seq) coupled with Nanopore sequencing to identify conditionally essential genes (CEGs) for the growth of <i>S. suis</i> P1/7 in active porcine serum (APS) and CSF derived from choroid plexus organoids. To our knowledge, this is the first successful application of ONT to Tn-library screening, enabling rapid local runs and a publicly available analysis pipeline. Through comparative fitness analyses, we identified 33 CEGs that support growth in APS and 25 CEGs in CS2026-01-01T00:00:00Z2026 Dec2026-06-06T04:03:01.163Z2026-05-25T03:11:59.348ZS-EPMC127075164139600710.1080/21505594.2025.2600145