UNLABELLED: The paper presents the study of a set of isolates of Streptococcus pneumoniae, which comprised two heterogeneous subpopulations, one of which was susceptible and the other resistant to optochin. 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 atpC gene and in one isolate in the atpA gene. One of the mutations in the atpC gene has not yet been published in the literature; it is a mutation at position 143T > 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 atpA and atpC genes. In the remaining two isolates, no clear genetic explanation for the optochin resistance in S. pneumoniae was found, based on current knowledge. IMPORTANCE: Globally, among the most fundamental tests used for the identification of Streptococcus pneumoniae 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 S. pneumoniae. This study compares whole-genome sequencing data of optochin-susceptible and optochin-resistant subpopulations of S. pneumoniae isolates and investigates the genetic probable causes of resistance in the genomes of optochin-resistant subpopulations.

3rd Faculty of Medicine Charles University Prague Czech Republic

The National Reference Laboratory for Meningococcal Infections Department of Air Borne Bacterial Infections Centre for Epidemiology and Microbiology National Institute of Public Health Prague Czech Republic

The National Reference Laboratory for Streptococcal Infections Department of Air Borne Bacterial Infections Centre for Epidemiology and Microbiology National Institute of Public Health Prague Czech Republic

Zobrazit více v PubMed

Werno AM, Murdoch DR. 2008. Medical microbiology: laboratory diagnosis of invasive pneumococcal disease. Clin Infect Dis 46:926–932. doi:10.1086/528798 PubMed DOI

Park HK, Lee SJ, Yoon JW, Shin JW, Shin HS, Kook JK, Myung SC, Kim W. 2010. Identification of the cpsA gene as a specific marker for the discrimination of Streptococcus pneumoniae from viridans group streptococci. J Med Microbiol 59:1146–1152. doi:10.1099/jmm.0.017798-0 PubMed DOI

Alberts B, Hopkin K, Johnson A, Morgan D, Roberts K, Walter P, Heald R. 2019. Essential cell biology. 5th ed, p 904. W. W. Norton & Company.

Fenoll A, Muñoz R, García E, de la Campa AG. 1994. Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniae Streptococcus oralis H+-ATPases. Mol Microbiol 12:587–598. doi:10.1111/j.1365-2958.1994.tb01045.x PubMed DOI

de la Campa AG, García E, Fenoll A, Muñoz R. 1997. Molecular bases of three characteristic phenotypes of pneumococcus: optochin-sensitivity, coumarin-sensitivity, and quinolone-resistance. Microb Drug Resist 3:177–193. doi:10.1089/mdr.1997.3.177 PubMed DOI

Morgenroth J, Levy R. 1911. Chemotherapie der Pneumokokeninfektion. Berl klin Wchnschr 48:1560.

Moore HF, Chesney AM. 1918. A further study of ethylhydrocuprein (optochin) in the treatment of acute lobar pneumonia. Arch Intern Med 19:659–681. doi:10.1001/archinte.1918.00090100096006 DOI

Moore HF. 1915. The action of ethylhydrocuprein (optochin) on type strains of pneumococci in vitro and in vivo, and on some other microorganisms in vitro. J Exp Med 22:269–285. doi:10.1084/jem.22.3.269 PubMed DOI PMC

Bowers EF, Jeffries LR. 1955. Optochin in the identification of str. pneumoniae. J Clin Pathol 8:58–60. doi:10.1136/jcp.8.1.58 PubMed DOI PMC

BOWEN MK, THIELE LC, STEARMAN BD, SCHAUB IG. 1957. The Optochin sensitivity test: a reliable method for identification of pneumococci. J Lab Clin Med 49:641–642. PubMed

Lund E. 1959. Diagnosis of pneumococci by the optochin and bile tests. Acta Pathol Microbiol Scand 47:308–315. doi:10.1111/j.1699-0463.1959.tb03720.x PubMed DOI

Kontiainen S, Sivonen A. 1987. Optochin resistance in Streptococcus pneumoniae strains isolated from blood and middle ear fluid. Eur J Clin Microbiol 6:422–424. doi:10.1007/BF02013101 PubMed DOI

Phillips G, Barker R, Brogan O. 1988. Optochin-resistant Streptococcus pneumoniae. Lancet 332:281. doi:10.1016/S0140-6736(88)92573-1 PubMed DOI

Vacková Z, Klímová M, Kozáková J. 2013. Ročník 22. Nová molekulární metoda a schéma typizace Streptococcus pneumoniae v České republice, Vol. 1, p 16–18. Zprávy Epidemiologie a Mikrobiologie. PubMed

Zerbino DR. 2010. Using the Velvet de novo assembler for short-read sequencing technologies. Curr Protoc Bioinformatics Chapter 11:Unit. doi:10.1002/0471250953.bi1105s31 PubMed DOI PMC

Jolley KA, Bray JE, Maiden MCJ. 2018. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res 3:124. doi:10.12688/wellcomeopenres.14826.1 PubMed DOI PMC

Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95:3140–3145. doi:10.1073/pnas.95.6.3140 PubMed DOI PMC

Enright MC, Spratt BG. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology (Reading) 144:3049–3060. doi:10.1099/00221287-144-11-3049 PubMed DOI

Jolley KA, Chan M-S, Maiden MCJ. 2004. mlstdbNet – distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 5:86. doi:10.1186/1471-2105-5-86 PubMed DOI PMC

Jolley KA, Bliss CM, Bennett JS, Bratcher HB, Brehony C, Colles FM, Wimalarathna H, Harrison OB, Sheppard SK, Cody AJ, Maiden MCJ. 2012. Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain. Microbiology (Reading) 158:1005–1015. doi:10.1099/mic.0.055459-0 PubMed DOI PMC

Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, Dempsey DM, Dickerman A, Dietrich EM, Kenyon RW, et al. . 2023. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res 51:D678–D689. doi:10.1093/nar/gkac1003 PubMed DOI PMC

Li H. 2013. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv. doi:10.48550/arXiv.1303.3997 DOI

Garrison E, Marth G. 2012. Haplotype-based variant detection from short-read sequencing. arXiv. doi:10.48550/arXiv.1207.3907 DOI

Dias CA, Agnes G, Frazzon APG, Kruger FD, d’Azevedo PA, Carvalho M da GS, Facklam RR, Teixeira LM. 2007. Diversity of mutations in the atpC gene coding for the c subunit of F0F1 ATPase in clinical isolates of optochin-resistant Streptococcus pneumoniae from Brazil. J Clin Microbiol 45:3065–3067. doi:10.1128/JCM.00891-07 PubMed DOI PMC

Nunes S, Sá-Leão R, de Lencastre H. 2008. Optochin resistance among Streptococcus pneumoniae strains colonizing healthy children in Portugal. J Clin Microbiol 46:321–324. doi:10.1128/JCM.02097-07 PubMed DOI PMC

Ikryannikova LN, Ischenko DS, Lominadze GG, Kanygina AV, Karpova IY, Kostryukova ES, Mayansky NA, Skvortsov VS, Ilina EN, Govorun VM. 2016. The mystery of the fourth clone: comparative genomic analysis of four non-typeable Streptococcus pneumoniae strains with different susceptibilities to optochin. Eur J Clin Microbiol Infect Dis 35:119–130. doi:10.1007/s10096-015-2516-5 PubMed DOI

Pinto TCA, Souza ARV, de Pina S, Costa NS, Borges Neto AA, Neves FPG, Merquior VLC, Dias CAG, Peralta JM, Teixeira LM. 2013. Phenotypic and molecular characterization of optochin-resistant Streptococcus pneumoniae isolates from Brazil, with description of five novel mutations in the ATPC gene. J Clin Microbiol 51:3242–3249. doi:10.1128/JCM.01168-13 PubMed DOI PMC

Ktari S, Ben Ayed NEH, Maalej S, Mnif B, Rhimi F, Hammami A. 2021. Clinical optochin resistant Streptococcus pneumoniae and Streptococcus pseudopneumoniae strains in Tunisia. J Infect Dev Ctries 15:672–677. doi:10.3855/jidc.13106 PubMed DOI

Raddaoui A, Ben Tanfous F, Achour W, Baaboura R, Ben Hassen A. 2018. Description of a novel mutation in the atpC gene in optochin-resistant Streptococcus pneumoniae strains isolates from Tunisia. Int J Antimicrob Agents 51:803–805. doi:10.1016/j.ijantimicag.2017.12.029 PubMed DOI

Cogné N, Claverys J, Denis F, Martin C. 2000. A novel mutation in the α-helix 1 of the C subunit of the F1/F0 ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate. Diagn Microbiol Infect Dis 38:119–121. doi:10.1016/s0732-8893(00)00182-6 PubMed DOI

Pikis A, Campos JM, Rodriguez WJ, Keith JM. 2001. Optochin resistance in Streptococcus pneumoniae: mechanism, significance, and clinical implications. J Infect Dis 184:582–590. doi:10.1086/322803 PubMed DOI

Nagata M, Ueda O, Shobuike T, Muratani T, Aoki Y, Miyamoto H. 2012. Emergence of optochin resistance among Streptococcus pneumoniae in Japan. Open J Med Microbiol 2. doi:10.4236/ojmm.2012.21002 DOI

Souza ARV, de Pina S, Costa NS, Neves FPG, Merquior VLC, Peralta JM, Pinto TCA, Teixeira LM. 2021. Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil. Sci Rep 11:7936. doi:10.1038/s41598-021-87071-8 PubMed DOI PMC

Cortes PR, Orio AGA, Regueira M, Piñas GE, Echenique J. 2008. Characterization of in vitro-generated and clinical optochin-resistant strains of Streptococcus pneumoniae isolated from Argentina. J Clin Microbiol 46:1930–1934. doi:10.1128/JCM.02318-07 PubMed DOI PMC

Cortes PR, Piñas GE, Albarracin Orio AG, Echenique JR. 2008. Subinhibitory concentrations of penicillin increase the mutation rate to optochin resistance in Streptococcus pneumoniae. J Antimicrob Chemother 62:973–977. doi:10.1093/jac/dkn322 PubMed DOI

Kozáková J, Vohrnová S, Okonji Z, Křížová P. 2024. Invazivní pneumokokové onemocnění v České republice v roce 2023. Zprávy CEM (SZÚ, Praha) 33:193–197.

Yunck R, Cho H, Bernhardt TG. 2016. Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria. Mol Microbiol 99:700–718. doi:10.1111/mmi.13258 PubMed DOI PMC

Tsui H-C, Zheng JJ, Magallon AN, Ryan JD, Yunck R, Rued BE, Bernhardt TG, Winkler ME. 2016. Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39. Mol Microbiol 100:1039–1065. doi:10.1111/mmi.13366 PubMed DOI PMC

Kaiser JC, Heinrichs DE. 2018. Branching out: alterations in bacterial physiology and virulence due to branched-chain amino acid deprivation. mBio 9:e01188-18. doi:10.1128/mBio.01188-18 PubMed DOI PMC

Basavanna S, Khandavilli S, Yuste J, Cohen JM, Hosie AHF, Webb AJ, Thomas GH, Brown JS. 2009. Screening of Streptococcus pneumoniae ABC transporter mutants demonstrates that LivJHMGF, a branched-chain amino acid ABC transporter, is necessary for disease pathogenesis. Infect Immun 77:3412–3423. doi:10.1128/IAI.01543-08 PubMed DOI PMC

Akhtar AA, Turner DP. 2022. The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: therapeutic and vaccine potential. Microb Pathog 171:105734. doi:10.1016/j.micpath.2022.105734 PubMed DOI

Hürlimann LM, Corradi V, Hohl M, Bloemberg GV, Tieleman DP, Seeger MA. 2016. The heterodimeric ABC transporter EfrCD mediates multidrug efflux in Enterococcus faecalis. Antimicrob Agents Chemother 60:5400–5411. doi:10.1128/AAC.00661-16 PubMed DOI PMC

Fox EJ, Reid-Bayliss KS, Emond MJ, Loeb LA. 2014. Accuracy of next generation sequencing platforms. Next Gener Seq Appl 1:1000106. doi:10.4172/jngsa.1000106 PubMed DOI PMC

Kellogg JA, Bankert DA, Elder CJ, Gibbs JL, Smith MC. 2001. Identification of Streptococcus pneumoniae revisited. J Clin Microbiol 39:3373–3375. doi:10.1128/JCM.39.9.3373-3375.2001 PubMed DOI PMC

Yahiaoui RY, den Heijer CDJ, Wolfs P, Bruggeman CA, Stobberingh EE. 2016. Evaluation of phenotypic and molecular methods for identification of Streptococcus pneumoniae. Future Microbiol 11:43–50. doi:10.2217/fmb.15.124 PubMed DOI