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Antimicrobial Agents and Chemotherapy, October 2009, p. 4211-4216, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00385-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Simple, Rapid, and Inexpensive Detection of Neisseria gonorrhoeae Resistance Mechanisms Using Heat-Denatured Isolates and SYBR Green-Based Real-Time PCR{triangledown}

Gayle Kugelman,1,2,3 John W. Tapsall,4 Namraj Goire,1,2,3 Melanie W. Syrmis,1,2,3 Athena Limnios,4 Stephen B. Lambert,1,2,3 Michael D. Nissen,1,2,3,5,6 Theo P. Sloots,1,2,3,5,6 and David M. Whiley1,2,3*

Queensland Children's Medical Research Institute, Infectious Diseases Laboratory, Royal Children's Hospital, Herston Road, Herston, QLD 4029, Queensland Australia,1 Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Herston, Queensland, Australia,2 Clinical Medical Virology Centre, University of Queensland, Queensland, Australia,3 WHO Collaborating Centre for STD and HIV, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales, Australia,4 Microbiology Division, Pathology Queensland, Royal Brisbane Hospital Campus, Brisbane, Queensland, Australia,5 Department of Paediatric and Child Health, University of Queensland, Brisbane, Queensland, Australia6

Received 22 March 2009/ Returned for modification 28 April 2009/ Accepted 6 June 2009

Neisseria gonorrhoeae has developed resistance to multiple classes of antimicrobials. There is now growing concern that without the availability of appropriate public health strategies to combat this problem, gonorrhea could become untreatable. For this reason, surveillance for gonococcal antimicrobial resistance must be optimal both in terms of obtaining a representative sample of gonococcal isolates and in terms of having the appropriate tools to identify resistance. To aid with this surveillance, molecular tools are increasingly being used. In the present study, we investigated the use of a simple heat denaturation protocol for isolate DNA preparation combined with SYBR green-based real-time PCR for the identification of mutations associated with N. gonorrhoeae antimicrobial resistance. A total of 109 clinical gonococcal isolates were tested by high-resolution melting (HRM) curve analysis for chromosomal mutations associated with gonococcal resistance to beta-lactam antibiotics: a penA 345A insertion, ponA L421P, mtrR G45D, substitutions at positions 120 and 121 in porB1b, and an adenine deletion in the mtrR promoter. An allele-specific PCR assay was also investigated for its ability to detect the adenine deletion in the mtrR promoter. The results were compared to those obtained by DNA sequencing. Our HRM assays provided the accurate discrimination of heat-treated isolates in which the sequence types differed in GC content, including isolates with the penA 345A insertion and the ponA L421P and mtrR G45D mutations. The allele-specific PCR assay accurately identified isolates with the adenine deletion in the mtrR promoter. Heat-denatured DNA combined with SYBR green-based real-time PCR offers a simple, rapid, and inexpensive means of detecting gonococcal resistance mechanisms. These methods may have broader application in the detection of polymorphisms associated with phenotypes of interest.

* Corresponding author. Mailing address: Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital & Health Service District, Herston Road, Herston, Queensland 4029, Australia. Phone: 61-7-3636 1623. Fax: 61-7-3636 1401. E-mail: d.whiley{at}uq.edu.au

{triangledown} Published ahead of print on 15 June 2009.

Antimicrobial Agents and Chemotherapy, October 2009, p. 4211-4216, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00385-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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