1887

Abstract

Purpose. Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without bla CTX-M-3, isolated from a patient.

Methodology. Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes.

Results. The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with bla CTX-M-3 failed to transfer bla CTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without bla CTX-M-3 experimentally received the plasmid encoding bla SHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2.

Conclusion. Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000634
2017-11-16
2019-08-22
Loading full text...

Full text loading...

/deliver/fulltext/jmm/67/1/33.html?itemId=/content/journal/jmm/10.1099/jmm.0.000634&mimeType=html&fmt=ahah

References

  1. Proctor RA, von Eiff C, Kahl BC, Becker K, Mcnamara P et al. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 2006;4:295–305 [CrossRef][PubMed]
    [Google Scholar]
  2. Melter O, Radojevič B. Small colony variants of Staphylococcus aureus—review. Folia Microbiol 2010;55:548–558 [CrossRef][PubMed]
    [Google Scholar]
  3. Sendi P, Proctor RA. Staphylococcus aureus as an intracellular pathogen: the role of small colony variants. Trends Microbiol 2009;17:54–58 [CrossRef][PubMed]
    [Google Scholar]
  4. Roggenkamp A, Sing A, Hornef M, Brunner U, Autenrieth IB et al. Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli. J Clin Microbiol 1998;36:2530–2534[PubMed]
    [Google Scholar]
  5. Gilligan PH, Gage PA, Welch DF, Muszynski MJ, Wait KR. Prevalence of thymidine-dependent Staphylococcus aureus in patients with cystic fibrosis. J Clin Microbiol 1987;25:1258–1261[PubMed]
    [Google Scholar]
  6. Besier S, Zander J, Siegel E, Saum SH, Hunfeld KP et al. Thymidine-dependent Staphylococcus aureus small-colony variants: human pathogens that are relevant not only in cases of cystic fibrosis lung disease. J Clin Microbiol 2008;46:3829–3832 [CrossRef][PubMed]
    [Google Scholar]
  7. Maduka-Ezeh A, Seville MT, Kusne S, Vikram HR, Blair JE et al. Thymidine auxotrophic Staphylococcus aureus small-colony variant endocarditis and left ventricular assist device infection. J Clin Microbiol 2012;50:1102–1105 [CrossRef][PubMed]
    [Google Scholar]
  8. Haltiner RC, Migneault PC, Robertson RG. Incidence of thymidine-dependent enterococci detected on Mueller-Hinton agar with low thymidine content. Antimicrob Agents Chemother 1980;18:365–368 [CrossRef][PubMed]
    [Google Scholar]
  9. Barker J, Healing D, Hutchison JG. Characteristics of some co-trimoxazole-resistant Enterobacteriaceae from infected patients. J Clin Pathol 1972;25:1086–1088 [CrossRef][PubMed]
    [Google Scholar]
  10. Tanner EI, Bullin CH. Thymidine-dependent Escherichia coli infection and some associated laboratory problems. J Clin Pathol 1974;27:565–568 [CrossRef][PubMed]
    [Google Scholar]
  11. King CH, Shlaes DM, Dul MJ. Infection caused by thymidine-requiring, trimethoprim-resistant bacteria. J Clin Microbiol 1983;18:79–83[PubMed]
    [Google Scholar]
  12. Kriegeskorte A, Block D, Drescher M, Windmüller N, Mellmann A et al. Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression. MBio 2014;5:e0144701414 [CrossRef][PubMed]
    [Google Scholar]
  13. Zander J, Besier S, Saum SH, Dehghani F, Loitsch S et al. Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus. Infect Immun 2008;76:1333–1339 [CrossRef][PubMed]
    [Google Scholar]
  14. Besier S, Ludwig A, Ohlsen K, Brade V, Wichelhaus TA. Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus. Int J Med Microbiol 2007;297:217–225 [CrossRef][PubMed]
    [Google Scholar]
  15. Chatterjee I, Kriegeskorte A, Fischer A, Deiwick S, Theimann N et al. In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of Staphylococcus aureus. J Bacteriol 2008;190:834–842 [CrossRef][PubMed]
    [Google Scholar]
  16. Besier S, Zander J, Kahl BC, Kraiczy P, Brade V et al. The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates. Antimicrob Agents Chemother 2008;52:2183–2189 [CrossRef][PubMed]
    [Google Scholar]
  17. Kahl BC, Belling G, Reichelt R, Herrmann M, Proctor RA et al. Thymidine-dependent small-colony variants of Staphylococcus aureus exhibit gross morphological and ultrastructural changes consistent with impaired cell separation. J Clin Microbiol 2003;41:410–413 [CrossRef][PubMed]
    [Google Scholar]
  18. Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008;8:159–166 [CrossRef][PubMed]
    [Google Scholar]
  19. Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 2005;18:657–686 [CrossRef][PubMed]
    [Google Scholar]
  20. Ronde-Oustau C, Lustig S, Dupieux C, Ferry T.Lyon BJI Study group Implant-associated ESBL-Klebsiella pneumonia producing small colony variant bone and joint infection in a healthy 40-year-old man. BMJ Case Rep 2017; doi:10.1136/bcr-2016-217542 [CrossRef][PubMed]
    [Google Scholar]
  21. Neilan BA, Jacobs D, del Dot T, Blackall LL, Hawkins PR et al. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int J Syst Bacteriol 1997;47:693–697 [CrossRef][PubMed]
    [Google Scholar]
  22. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–2239[PubMed]
    [Google Scholar]
  23. Rasheed JK, Anderson GJ, Yigit H, Queenan AM, Doménech-Sánchez A et al. Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18. Antimicrob Agents Chemother 2000;44:2382–2388 [CrossRef][PubMed]
    [Google Scholar]
  24. Shibata N, Kurokawa H, Doi Y, Yagi T, Yamane K et al. PCR classification of CTX-M-type beta-lactamase genes identified in clinically isolated gram-negative bacilli in Japan. Antimicrob Agents Chemother 2006;50:791–795 [CrossRef][PubMed]
    [Google Scholar]
  25. Yagi T, Kurokawa H, Shibata N, Shibayama K, Arakawa Y. A preliminary survey of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Japan. FEMS Microbiol Lett 2000;184:53–56[PubMed]
    [Google Scholar]
  26. Dutour C, Bonnet R, Marchandin H, Boyer M, Chanal C et al. CTX-M-1, CTX-M-3, and CTX-M-14 beta-lactamases from Enterobacteriaceae isolated in France. Antimicrob Agents Chemother 2002;46:534–537 [CrossRef][PubMed]
    [Google Scholar]
  27. Horiuchi K, Matsumoto T, Ota Y, Kasuga E, Negishi T et al. Addition of thymidine to culture media for accurate examination of thymidine-dependent small-colony variants of methicillin-resistant Staphylococcus aureus: a pilot study. J Microbiol Methods 2015;110:40–44 [CrossRef][PubMed]
    [Google Scholar]
  28. Yagi T, Kurokawa H, Senda K, Ichiyama S, Ito H et al. Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes. Antimicrob Agents Chemother 1997;41:2606–2611[PubMed]
    [Google Scholar]
  29. Zhang J, Keller K, Takemoto JY, Bensaci M, Litke A et al. Synthesis and combinational antibacterial study of 5''-modified neomycin. J Antibiot 2009;62:539–544 [CrossRef][PubMed]
    [Google Scholar]
  30. Gniadkowski M, Schneider I, Pałucha A, Jungwirth R, Mikiewicz B et al. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob Agents Chemother 1998;42:827–832[PubMed]
    [Google Scholar]
  31. Matthews DA, Appelt K, Oatley SJ, Xuong NH. Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate. J Mol Biol 1990;214:923–936 [CrossRef][PubMed]
    [Google Scholar]
  32. Matthews DA, Villafranca JE, Janson CA, Smith WW, Welsh K et al. Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. J Mol Biol 1990;214:937–948 [CrossRef][PubMed]
    [Google Scholar]
  33. Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EP, de La Cruz F. Mobility of plasmids. Microbiol Mol Biol Rev 2010;74:434–452 [CrossRef][PubMed]
    [Google Scholar]
  34. Precit MR, Wolter DJ, Griffith A, Emerson J, Burns JL et al. Optimized in vitro antibiotic susceptibility testing method for small-colony variant Staphylococcus aureus. Antimicrob Agents Chemother 2016;60:1725–1735 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000634
Loading
/content/journal/jmm/10.1099/jmm.0.000634
Loading

Data & Media loading...

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error