Identification of Single Nucleotide Polymorphisms in Neisseria Gonorrhoeae Isolates with Elevated Cephalosporin MICs Using Next Generation Whole Genome Sequencing

Links, Philip

Thursday, June 12, 2014: 8:40 AM

Dogwood A

Philip Links, PhD MSc MPA¹, Irene Martin, BSc², Matthew Links, PhD³, Qing Liu, PhD⁴, Ping Yan, PhD⁵, Jun Wu, PhD⁵ and Tom Wong, MD, MPH, FRCPC⁶, 1Professional Guidelines and Public Health Practice Division, Center for Communicable Diseases and Infection Control, Infectious Disease Prevention and Control Branch, Public Health Agency of Canada, Ottawa, ON, Canada, 2Streptococcus and STI Unit, National Microbiology Laboratory, Winnipeg, MB, Canada, 3Bio-Products and Bioprocessing, National Science Program, Science and Technology Branch, Agriculture and Agri-Food Canada, Agriculture and Agri-Food Canada, Saskatoon, SK, Canada, 4Translational Biosciences, Human Health and Therapeutics Portfolio, National Research Council of Canada, National Research Council of Canada, Ottawa, ON, Canada, 5Professional Guidelines and Public Health Practice Division, Center for Communicable Diseases and Infection Control, Infectious Disease Prevention and Control Branch, Public Health Agency of Canada, Public Health Agency of Canada, Ottawa, ON, Canada, 6Professional Guidelines and Public Health Practice Division, Centre for Communicable Diseases and Infection Control, Public Health Agency of Canada, Ottawa, ON, Canada

Background: Neisseria gonorrhoeae (NG) remains the second most common bacterial sexually transmitted disease in Canada and the USA. If untreated, infections can result in serious injury and in some cases infertility. Diagnosis of NG infection is frequently performed by nucleic acid amplification tests which provide no information about antibiotic resistance. Emergence of NG strains with higher minimum inhibitory concentrations (MICs) to third generation cephalosporins, (cefixime, ceftriaxone) and azithromycin is threatening last available treatment options.

Methods: MiSeq whole genome sequencing and bioinformatics were used to detect single nucleotide polymorphisms (SNPs) present in 200 NG samples. Isolates with low (<0.032 μg/ml) and high (≥0.125 μg/ml) MICs to cefixime, and low (<0.016 μg/ml) and high (≥0.125 μg/ml) MICs to ceftriaxone were grouped for further analysis. In-silico assay validation using two tailed student t-testing, and the power of the test showed low and high MIC groups could be distinguished. The genes where SNPs were located were blinded until assays were optimized.

Results: Sequencing data was evaluated for both cefixime and ceftriaxone isolates and 12 SNPs with the greatest difference between low and high MIC groups were determined. Predicted sensitivities of 12 SNP cefixime and ceftriaxone assays were calculated to be 97% (58/60), and 94% (49/52). The specificities of cefixime and ceftriaxone assays were 83% (68/82) and 89% (51/57). Some informative nonsynonymous SNPs were located in genes including efflux pumps, proteins involved in ribosomal function and genes known to confer antibiotic resistance.

Conclusions: Novel SNPs can be used to distinguish NG strains with high cephalosporin MICs from low MICs and are being used for the development of diagnostic tests to identify antimicrobial resistance in N. gonorrhoea. New rapid genotypic resistance assays could inform appropriate use of antibiotic treatment for gonorrhea infections, lowering the risk of treatment failure, and reducing the spread of multidrug resistant gonorrhea.

5B 5 Identification of Single Nucleotide Polymorphisms in Neisseria Gonorrhoeae Isolates with Elevated Cephalosporin MICs Using Next Generation Whole Genome Sequencing