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Antimicrobial Agents and Chemotherapy, May 2009, p. 2020-2027, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01349-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Concurrent Titration and Determination of Antibiotic Resistance in Ureaplasma Species with Identification of Novel Point Mutations in Genes Associated with Resistance{triangledown} ,{dagger}

Michael L. Beeton,1 Victoria J. Chalker,2 Nicola C. Maxwell,1 Sailesh Kotecha,1 and O. Brad Spiller1*

Cardiff University, School of Medicine, Department of Child Health, University Hospital of Wales, Heath Park, Cardiff CF14 4XN, United Kingdom,1 Respiratory and Systemic Infections Laboratory, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, United Kingdom2

Received 8 October 2008/ Returned for modification 2 December 2008/ Accepted 25 February 2009

Antibiotic resistance determination of Ureaplasma spp. (Ureaplasma parvum and Ureaplasma urealyticum) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma-positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.

* Corresponding author. Mailing address: Cardiff University, School of Medicine, Dept. of Child Health, 5th Floor, University Hospital of Wales, Heath Park, Cardiff CF14 4XN, United Kingdom. Phone: 44 (0)2920 742394. Fax: 44 (0)2920 744283. E-mail: SpillerB{at}cf.ac.uk

{triangledown} Published ahead of print on 9 March 2009.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, May 2009, p. 2020-2027, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01349-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Beeton, M. L., Chalker, V. J., Kotecha, S., Spiller, O. B. (2009). Comparison of full gyrA, gyrB, parC and parE gene sequences between all Ureaplasma parvum and Ureaplasma urealyticum serovars to separate true fluoroquinolone antibiotic resistance mutations from non-resistance polymorphism. J Antimicrob Chemother 64: 529-538 [Abstract] [Full Text]  


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