Antimicrobial Resistance Screening in Chlamydia trachomatis by Optimized McCoy Cell Culture System and Direct qPCR-Based Monitoring of Chlamydial Growth

  • Tomislav Meštrović
  • Dezső P. Virok
  • Sunčanica Ljubin-Sternak
  • Tímea Raffai
  • Katalin Burián
  • Jasmina Vraneš
Part of the Methods in Molecular Biology book series (MIMB, volume 2042)


Obligate intracellular localization of Chlamydia trachomatis (C. trachomatis) complicates antimicrobial sensitivity testing efforts that we are so accustomed to in routine bacteriology. Cell culture systems with immunofluorescence staining, to identify cellular inclusions in the presence of various concentrations of antimicrobial drugs, are still the most pervasive techniques, but more specific and sensitive nucleic acid concentration measuring methods are increasingly being used. Here we describe how to approach antimicrobial susceptibility/resistance screening in C. trachomatis by using a McCoy cell culture system, optimized by a research group from Croatia, and direct qPCR-based monitoring of chlamydial growth, optimized by a research group from Hungary.

Key words

Chlamydia trachomatis Antimicrobial susceptibility testing Antimicrobial resistance Cell culture qPCR 



Tomislav Meštrović and Dezső P. Virok contributed equally to this work.


  1. 1.
    Suchland RJ, Geisler WM, Stamm WE (2003) Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp. Antimicrob Agents Chemother 47:636–642CrossRefGoogle Scholar
  2. 2.
    Meštrović T, Ljubin-Sternak S (2018) Molecular mechanisms of Chlamydia trachomatis resistance to antimicrobial drugs. Front Biosci (Landmark Ed) 23:656–670CrossRefGoogle Scholar
  3. 3.
    Ljubin-Sternak S, Meštrović T (2014) Chlamydia trachomatis and genital mycoplasmas: pathogens with an impact on human reproductive health. J Pathog 2014:183167Google Scholar
  4. 4.
    Ljubin-Sternak S, Meštrović T, Vilibić-Čavlek T, Mlinarić-Galinović G, Sviben M, Markotić A, Škerk V (2013) In vitro susceptibility of urogenital Chlamydia trachomatis strains in a country with high azithromycin consumption rate. Folia Microbiol (Praha) 58:361–365CrossRefGoogle Scholar
  5. 5.
    Meštrović T, LjubinSternak S, Bedenić B (2015) Technical aspects of Chlamydia trachomatis antimicrobial susceptibility testing in cell culture system. Technical Journal 9:136–141Google Scholar
  6. 6.
    Meštrović T, Ljubin-Sternak S, Sviben M, Bedenić B, Vraneš J, Markotić A, Škerk V (2016) Antimicrobial sensitivity profile of Chlamydia trachomatis isolates from Croatia in McCoy cell culture system and comparison with the literature. Clin Lab 62:357–364CrossRefGoogle Scholar
  7. 7.
    Samra Z, Rosenberg S, Soffer Y, Dan M (2001) In vitro susceptibility of recent clinical isolates of Chlamydia trachomatis to macrolides and tetracyclines. Diagn Microbiol Infect Dis 39:177–179CrossRefGoogle Scholar
  8. 8.
    Donati M, Di Francesco A, D’Antuono A, Delucca F, Shurdhi A, Moroni A, Baldelli R, Cevenini R (2010) In vitro activities of several antimicrobial agents against recently isolated and genotyped Chlamydia trachomatis urogenital serovars D through K. Antimicrob Agents Chemother 54:5379–5380CrossRefGoogle Scholar
  9. 9.
    Bhengraj AR, Vardhan H, Srivastava P, Salhan S, Mittal A (2010) Decreased susceptibility to azithromycin and doxycycline in clinical isolates of Chlamydia trachomatis obtained from recurrently infected female patients in India. Chemotherapy 56:371–377CrossRefGoogle Scholar
  10. 10.
    Kai S, Wada K, Sadahira T, Araki M, Ishii A, Watanabe T, Monden K, Uno S, Araki T, Nasu Y (2017) Antimicrobial susceptibilities of Chlamydia trachomatis isolated from the urethra and pharynx of Japanese males. J Infect Chemother 23:512–516CrossRefGoogle Scholar
  11. 11.
    Osaka I, Hills JM, Kieweg SL, Shinogle HE, Moore DS, Hefty PS (2012) An automated image-based method for rapid analysis of Chlamydia infection as a tool for screening antichlamydial agents. Antimicrob Agents Chemother 56:4184–4188CrossRefGoogle Scholar
  12. 12.
    Southern T, Bess L, Harmon J, Taylor L, Caldwell H (2012) Fluorometric high-throughput assay for measuring chlamydial neutralizing antibody. Clin Vaccine Immunol 19:1864–1869CrossRefGoogle Scholar
  13. 13.
    Bogdanov A, Endrész V, Urbán S, Lantos I, Deák J, Burián K, Önder K, Ayaydin F, Balázs P, Virok DP (2014) Application of DNA chip scanning technology for automatic detection of Chlamydia trachomatis and Chlamydia pneumoniae inclusions. Antimicrob Agents Chemother 58:405–413CrossRefGoogle Scholar
  14. 14.
    Peuchant O, Duvert JP, Clerc M, Raherison S, Bébéar C, Bébéar CM, de Barbeyrac B (2011) Effects of antibiotics on Chlamydia trachomatis viability as determined by real-time quantitative PCR. J Med Microbiol 60:508–514CrossRefGoogle Scholar
  15. 15.
    Eszik I, Lantos I, Önder K, Somogyvári F, Burián K, Endrész V, Virok DP (2016) High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells. J Microbiol Methods 120:15–22CrossRefGoogle Scholar
  16. 16.
    Párducz L, Eszik I, Wagner G, Burián K, Endrész V, Virok DP (2016) Impact of antiseptics on Chlamydia trachomatis growth. Lett Appl Microbiol 63:260–267CrossRefGoogle Scholar
  17. 17.
    Bogdanov A, Janovák L, Lantos I, Endrész V, Sebők D, Szabó T, Dékány I, Deák J, Rázga Z, Burián K, Virok DP (2017) Nonactivated titanium-dioxide nanoparticles promote the growth of Chlamydia trachomatis and decrease the antimicrobial activity of silver nanoparticles. J Appl Microbiol 123:1335–1345CrossRefGoogle Scholar
  18. 18.
    Belland RJ, Nelson DE, Virok D, Crane DD, Hogan D, Sturdevant D, Beatty WL, Caldwell HD (2003) Transcriptome analysis of chlamydial growth during IFN-gamma-mediated persistence and reactivation. Proc Natl Acad Sci U S A 100:15971–15976CrossRefGoogle Scholar
  19. 19.
    Atlas RM (ed) (2010) Handbook of microbiological media, 4th edn. CRC Press, LondonGoogle Scholar
  20. 20.
    Labiran C, Clarke IN, Cutcliffe LT, Wang Y, Skilton RJ, Persson K, Bjartling C, Herrmann B, Christerson L, Marsh P (2012) Genotyping markers used for multi locus VNTR analysis with ompA (MLVA-ompA) and multi sequence typing (MST) retain stability in Chlamydia trachomatis. Front Cell Infect Microbiol 2:68CrossRefGoogle Scholar
  21. 21.
    Barnes RC (1989) Laboratory diagnosis of human chlamydial infections. Clin Microbiol Rev 2:119–136CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Tomislav Meštrović
    • 1
    • 2
  • Dezső P. Virok
    • 3
  • Sunčanica Ljubin-Sternak
    • 4
    • 5
  • Tímea Raffai
    • 3
  • Katalin Burián
    • 3
  • Jasmina Vraneš
    • 4
    • 5
  1. 1.University North, University Centre VaraždinVaraždinCroatia
  2. 2.Clinical Microbiology and Parasitology UnitPolyclinic “Dr. Zora Profozić”ZagrebCroatia
  3. 3.Institute of Medical Microbiology and ImmunobiologyUniversity of SzegedSzegedHungary
  4. 4.Medical Microbiology Department, School of MedicineUniversity of ZagrebZagrebCroatia
  5. 5.Clinical Microbiology DepartmentTeaching Institute of Public Health “Dr. Andrija Štampar”ZagrebCroatia

Personalised recommendations