BACKGROUND: The resistance of Streptococcus pneumoniae to macrolides is becoming an increasingly important issue and thus it is important to understand the genetics related to adaptation of this species to the widespread use of antibiotics in Europe. The 58 isolates of S. pneumoniae belonging to sequence type (ST) 416 and serotype 19A and to several different phenotypes originated from Italy, Portugal and Czech Republic were thus sequenced on Illumina MiSeq. The aim of the study was to describe genetical origine of isolates, investigate their macrolide resistance and suggest reasons for spread of ST416 in the Czech Republic. RESULTS: Investigation of genes associated with serotype determined serotype switch between 15B and 19A serotypes and core genome multilocus sequence typing (cgMLST) confirmed the origine of concerned isolates in Netherlands15B-37 clone. Inspected genomes proved variability of genes associated with the macrolide resistance even within closely genetically relative isolates. CONCLUSIONS: Participation of 19A/ST416 on the spread of Netherlands15B-37 is accompanied by serotype switch between 19A and 15B serotypes and with acquisition of genes involved in macrolide resistance to the clone that was originally macrolide susceptible. There is evident tendency to interchanging and modifications of these and surrounding genes, that could lead to accelerate spreading of this sequence type in regions with high macrolide consumption.

Centre for Epidemiology and Microbiology National Institute of Public Health Prague Czech Republic

Department of Clinical Microbiology Faculty of Medicine and University Hospital Charles University Hradec Kralove Czech Republic

Department of Infectious Diseases Istituto Superiore di Sanità Rome Italy

Department of Laboratory Medicine 3rd Faculty of Medicine Charles University and Kralovske Vinohrady University Hospital Prague Czech Republic

Faculty of Medicine Biomedical Center Charles University Plzen Czech Republic

See more in PubMed

O’Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, McCall N, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet. 2009;374(9693):893–902. doi: 10.1016/S0140-6736(09)61204-6. PubMed DOI

Regev-Yochay G, Raz M, Dagan R, Porat N, Shainberg B, Pinco E, et al. Nasopharyngeal carriage of Streptococcus pneumoniae by adults and children in community and family settings. Clin Infect Dis. 2004;38(5):632–639. doi: 10.1086/381547. PubMed DOI

Bogaert D, de Groot R, Hermans PWM. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis. 2004;4(3):144–154. doi: 10.1016/S1473-3099(04)00938-7. PubMed DOI

Geno KA, Gilbert GL, Song JY, Skovsted IC, Klugman KP, Jones C, et al. Pneumococcal capsules and their types: past, present, and future. Clin Microbiol Rev. 2015;28(3):871–899. doi: 10.1128/Cmr.00024-15. PubMed DOI PMC

Aguiar SI, Pinto FR, Nunes S, Serrano I, Melo-Cristino J, Sa-Leao R, et al. Denmark (14)-230 clone as an increasing cause of pneumococcal infection in Portugal within a background of diverse serotype 19A lineages. J Clin Microbiol. 2010;48(1):101–108. doi: 10.1128/Jcm.00665-09. PubMed DOI PMC

Moore MR, Gertz RE, Woodbury RL, Barkocy-Gallagher GA, Schaffner W, Lexau C, et al. Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. J Infect Dis. 2008;197(7):1016–1027. doi: 10.1086/528996. PubMed DOI

Del Grosso M, Camilli R, D'Ambrosio F, Petrucci G, Melchiorre S, Moschioni M, et al. Increase of pneumococcal serotype 19A in Italy is due to expansion of the piliated clone ST416/CC199. J Med Microbiol. 2013;62:1220–1225. doi: 10.1099/jmm.0.061242-0. PubMed DOI

van der Linden M, Reinert RR, Kern WV, Imohl M. Epidemiology of serotype 19A isolates from invasive pneumococcal disease in German children. BMC Infect Dis. 2013;13:70. doi: 10.1186/1471-2334-13-70. PubMed DOI PMC

Kaur R, Casey JR, Pichichero ME. Emerging Streptococcus pneumoniae strains colonizing the nasopharynx in children after 13-valent pneumococcal conjugate vaccination in comparison to the 7-valent era, 2006-2015. Pediatr Infect Dis J. 2016;35(8):901–906. doi: 10.1097/Inf.0000000000001206. PubMed DOI PMC

Domingues CMAS, Verani JR, Renoiner EIM, Brandileone MCD, Flannery B, de Oliveira LH, et al. Effectiveness of ten-valent pneumococcal conjugate vaccine against invasive pneumococcal disease in Brazil: a matched case-control study. Lancet Respir Med. 2014;2(6):464–471. doi: 10.1016/S2213-2600(14)70060-8. PubMed DOI PMC

Naucler P, Galanis I, Morfeldt E, Darenberg J, Ortqvist A, Henriques-Normark B. Comparison of the impact of pneumococcal conjugate vaccine 10 or pneumococcal conjugate vaccine 13 on invasive pneumococcal disease in equivalent populations. Clin Infect Dis. 2017;65(11):1780–1789. doi: 10.1093/cid/cix685. PubMed DOI PMC

Quirk SJ, Haraldsson G, Hjalmarsdottir MA, van Tonder AJ, Hrafnkelsson B, Bentley SD, et al. Vaccination of Icelandic children with the 10-valent pneumococcal vaccine leads to a significant herd effect among adults in Iceland. J Clin Microbiol. 2019;57(4):1. doi: 10.1128/JCM.01766-18. PubMed DOI PMC

Muñoz-Almagro C, Jordan I, Gene A, Latorre C, Garcia-Garcia JJ, Pallares R. Emergence of invasive pneumococcal disease caused by nonvaccine serotypes in the era of 7-valent conjugate vaccine. Clin Infect Dis. 2008;46(2):174–182. doi: 10.1086/524660. PubMed DOI

Tyrrell GJ. The changing epidemiology of Streptococcus pneumoniae serotype 19A clonal complexes. J Infect Dis. 2011;203(10):1345–1347. doi: 10.1093/infdis/jir056. PubMed DOI PMC

Deng X, Arya G, Memari N, Mackenzie R, MacMullin G, Low DE, et al. Genetic analysis of invasive pneumococcal isolates from children in Ontario, Canada, 2007-2012. Pediatr Infect Dis J. 2015;34(6):594–598. doi: 10.1097/INF.0000000000000697. PubMed DOI

Sadowy E, Kuch A, Gniadkowski M, Hryniewicz W. Expansion and evolution of the Streptococcus pneumoniae Spain9V-ST156 clonal complex in Poland. Antimicrob Agents Chemother. 2010;54(5):1720–1727. doi: 10.1128/AAC.01340-09. PubMed DOI PMC

Ramos V, Parra EL, Duarte C, Moreno J. Characterization of Streptococcus pneumoniae invasive serotype 19A isolates recovered in Colombia. Vaccine. 2014;32(7):755–758. doi: 10.1016/j.vaccine.2013.12.024. PubMed DOI

Mott MP, Caierão J, Cunha GR, Del Maschi MM, Pizzutti K, d’Azevedo P, et al. Emergence of serotype 19A Streptococcus pneumoniae after PCV10 associated with a ST320 in adult population, in Porto Alegre, Brazil. Epidemiol Infect. 2019;147:e93. doi: 10.1017/S0950268819000013. PubMed DOI PMC

Zhao C, Li Z, Zhang F, Zhang X, Ji P, Zeng J, et al. Serotype distribution and antibiotic resistance of Streptococcus pneumoniae isolates from 17 Chinese cities from 2011 to 2016. BMC Infect Dis. 2017;17(1):804. doi: 10.1186/s12879-017-2880-0. PubMed DOI PMC

Vancikova Z, Trojanek M, Zemlickova H, Blechova Z, Motlova J, Matejkova J, et al. Pneumococcal urinary antigen positivity in healthy colonized children: is it age dependent? Wien Klin Wochenschr. 2013;125(17–18):495–500. doi: 10.1007/s00508-013-0405-4. PubMed DOI

Kozakova J, Sebestova H, Krizova P. Invasive pneumococcal disease in the Czech Republic in 2015. Bull Centre Epidemiol Microbiol. 2016;25:100–107.

Zemlickova H, Malisova L, Spanelova P, Jakubu V, Kozakova J, Musilek M, et al. Molecular characterization of serogroup 19 Streptococcus pneumoniae in the Czech Republic in the post-vaccine era. J Med Microbiol. 2018;67:1003–1011. doi: 10.1099/jmm.0.000765. PubMed DOI PMC

Malisova L, Urbaskova P, Jakubu V, Spanelova P, Kozakova J, Musilek M, et al. Surveillance of antibiotic resistance of Streptococcus pneumoniae in the Czech Republic, respiratory study results, 2010–2017. Epidemiol Microbiol Immunol. 2019;2:75–81. PubMed

Public databases for molecular typing and microbial genome diversity. https://pubmlst.org/ Accessed January 2020.

Feil EJ, Smith JM, Enright MC, Spratt BG. Estimating recombinational parameters in Streptococcus pneumoniae from multilocus sequence typing data. Genetics. 2000;154(4):1439–1450. PubMed PMC

Beall BW, Gertz RE, Hulkower RL, Whitney CG, Moore MR, Brueggemann AB. Shifting genetic structure of invasive serotype 19A pneumococci in the United States. J Infect Dis. 2011;203(10):1360–1368. doi: 10.1093/infdis/jir052. PubMed DOI PMC

Croucher NJ, Hanage WP, Harris SR, McGee L, van der Linden M, de Lencastre H, et al. Variable recombination dynamics during the emergence, transmission and ‘disarming’ of a multidrug-resistant pneumococcal clone. BMC Biol. 2014;12:49. doi: 10.1186/1741-7007-12-49. PubMed DOI PMC

Hulten KG, Kaplan SL, Lamberth LB, Barson WJ, Romero JR, Lin PL, et al. Changes in Streptococcus pneumoniae serotype 19A invasive infections in children from 1993 to 2011. J Clin Microbiol. 2013;51(4):1294–1297. doi: 10.1128/JCM.00058-13. PubMed DOI PMC

Fenoll A, Granizo JJ, Giménez MJ, Yuste J, Aguilar L. Secular trends (1990-2013) in serotypes and associated non-susceptibility of S. pneumoniae isolates causing invasive disease in the pre−/post-era of pneumococcal conjugate vaccines in Spanish regions without universal paediatric pneumococcal vaccination. Vaccine. 2015;33(42):5691–5699. doi: 10.1016/j.vaccine.2015.08.009. PubMed DOI

Brueggemann AB, Pai R, Crook DW, Beall B. Vaccine escape recombinants emerge after pneumococcal vaccination in the United States. PLoS Pathog. 2007;3(11):1628–1636. doi: 10.1371/journal.ppat.0030168. PubMed DOI PMC

Gertz RE, McEllistrem MC, Boxrud DJ, Li ZY, Sakota V, Thompson TA, et al. Clonal distribution of invasive pneumococcal isolates from children and selected adults in the United States prior to 7-valent conjugate vaccine introduction. J Clin Microbiol. 2003;41(9):4194–4216. doi: 10.1128/Jcm.41.9.4194-4216.2003. PubMed DOI PMC

Gladstone RA, Devine V, Jones J, Cleary D, Jefferies JM, Bentley SD, et al. Pre-vaccine serotype composition within a lineage signposts its serotype replacement - a carriage study over 7 years following pneumococcal conjugate vaccine use in the UK. Microb Genomics. 2017;3(6):000119. doi: 10.1099/mgen.0.000119. PubMed DOI PMC

Makarewicz O, Lucas M, Brandt C, Herrmann L, Albersmeier A, Ruckert C, et al. Whole genome sequencing of 39 invasive Streptococcus pneumoniae sequence type 199 isolates revealed switches from serotype 19A to 15B. PLoS One. 2017;12(1):e0169370. doi: 10.1371/journal.pone.0169370. PubMed DOI PMC

ECDC. Antimicrobial consumption database (ESAC-Net). https://www.ecdc.europa.eu/en/antimicrobial-consumption/surveillance-and-disease-data/database Accessed Nov. 2019 2019.

de Vries LE, Christensen H, Agerso Y. The diversity of inducible and constitutively expressed erm (C) genes and association to different replicon types in staphylococci plasmids. Mob Genet Elem. 2012;2(2):72–80. doi: 10.4161/mge.20109. PubMed DOI PMC

Kataja J, Huovinen P, Seppala H, Grp MRS. Erythromycin resistance genes in group a streptococci of different geographical origins. J Antimicrob Chemother. 2000;46(5):789–792. doi: 10.1093/jac/46.5.789. PubMed DOI

Rudolph K, Bruce MG, Bulkow L, Zulz T, Reasonover A, Harker-Jones M, et al. Molecular epidemiology of serotype 19A Streptococcus pneumoniae among invasive isolates from Alaska, 1986–2010. Int J Circumpol Heal. 2013;72:611–617. doi: 10.3402/ijch.v72i0.20854. PubMed DOI PMC

Hasselmann C, European Soc Clinical Microbiology Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. Clin Microbiol Infect. 2003;9(8):1–7. PubMed

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Nurk S, Bankevich A, Antipov D, Gurevich A, Korobeynikov A, Lapidus A, et al. Assembling genomes and mini-metagenomes from highly chimeric reads. In: Deng M, Jiang R, Sun F, Zhang X, et al., editors. Research in computational molecular biology. Berlin, Heidelberg: Springer Berlin Heidelberg; 2013. pp. 158–170.

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–2069. doi: 10.1093/bioinformatics/btu153. PubMed DOI

Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20(1):238. doi: 10.1186/s13059-019-1832-y. PubMed DOI PMC

Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011;27(21):2987–2993. doi: 10.1093/bioinformatics/btr509. PubMed DOI PMC

Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, et al. The variant call format and VCFtools. Bioinformatics. 2011;27(15):2156–2158. doi: 10.1093/bioinformatics/btr330. PubMed DOI PMC