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The aim of the study was to compare the results of serotyping, multilocus sequence typing (MLST), ribosomal multilocus sequence typing (rMLST), and variation analysis of these subpopulations and to investigate the genetic probable causes of optochin resistance. The strains studied were cultured from samples taken from patients with invasive pneumococcal disease in the Czech Republic in 2019 and 2020. A total of 10 studied pairs of isolates were subject to serotyping and whole-genome sequencing (WGS). None of the typing methods (serotyping, MLST, or rMLST) applied to pairs of optochin-susceptible and optochin-resistant isolates revealed differences in serotype, sequence type, or ribosomal sequence type. The WGS data analysis identified point mutations in ATP (adenosine triphosphate) synthase genes in 8 of the 10 optochin-resistant isolates. In seven optochin-resistant isolates, the mutation was found in the\n              <jats:italic>atp<\/jats:italic>\n              C gene and in one isolate in the\n              <jats:italic>atp<\/jats:italic>\n              A gene. One of the mutations in the\n              <jats:italic>atp<\/jats:italic>\n              C gene has not yet been published in the literature; it is a mutation at position 143T &gt; C with an amino acid change of Val48Ala. In 8 out of the 10 optochin-resistant isolates, the possible genetic basis for resistance was identified, involving point mutations in the\n              <jats:italic>atp<\/jats:italic>\n              A and\n              <jats:italic>atp<\/jats:italic>\n              C genes. In the remaining two isolates, no clear genetic explanation for the optochin resistance in\n              <jats:italic>S. pneumoniae<\/jats:italic>\n              was found, based on current knowledge.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              Globally, among the most fundamental tests used for the identification of\n              <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n              isolates is determining susceptibility to optochin. In the last 2 decades, optochin-resistant strains have been frequently reported in the literature, which can lead to the misidentification of\n              <jats:italic>S. pneumoniae<\/jats:italic>\n              . This study compares whole-genome sequencing data of optochin-susceptible and optochin-resistant subpopulations of\n              <jats:italic>S. pneumoniae<\/jats:italic>\n              isolates and investigates the genetic probable causes of resistance in the genomes of optochin-resistant subpopulations.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.01939-24","type":"journal-article","created":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T14:09:33Z","timestamp":1740492573000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Comparison of\n            <i>Streptococcus pneumoniae<\/i>\n            isolates occurring in optochin-susceptible and optochin-resistant variants by analyzing whole-genome sequencing data"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7256-1642","authenticated-orcid":true,"given":"Sandra","family":"Vohrnov\u00e1","sequence":"first","affiliation":[{"name":"The National Reference Laboratory for Streptococcal Infections, Department of Air-Borne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health","place":["Prague, Czech Republic"]},{"name":"Third Faculty of Medicine, Charles University","place":["Prague, Czech Republic"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3923-8281","authenticated-orcid":false,"given":"Jana","family":"Koz\u00e1kov\u00e1","sequence":"additional","affiliation":[{"name":"The National Reference Laboratory for Streptococcal Infections, Department of Air-Borne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health","place":["Prague, Czech Republic"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1272-9304","authenticated-orcid":false,"given":"Michal","family":"Honskus","sequence":"additional","affiliation":[{"name":"Third Faculty of Medicine, Charles University","place":["Prague, Czech Republic"]},{"name":"The National Reference Laboratory for Meningococcal Infections, Department of Air-Borne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health","place":["Prague, Czech Republic"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1086\/528798"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.017798-0"},{"key":"e_1_3_4_4_2","first-page":"904","volume-title":"Essential cell biology","author":"Alberts B","year":"2019","unstructured":"Alberts B, Hopkin K, Johnson A, Morgan D, Roberts K, Walter P, HealdR. 2019. 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