1887

Abstract

Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, .

We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I (248) and GyrA D87N, D87Y and D87H (259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in .

Following assay validation, the 248 and 259 SYBR-MAMAs were tested on 84 Australian clinical isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing.

Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes.

Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Funding
This study was supported by the:
  • University of the Sunshine Coast
    • Principle Award Recipient: MaddenDanielle E.
  • Wishlist (Award 2019-14)
    • Principle Award Recipient: FraserTamieka A.
  • Wishlist (Award 2019-14)
    • Principle Award Recipient: PriceErin P.
  • Wishlist (Award 2019-14)
    • Principle Award Recipient: SarovichDerek S.
  • Wishlist (Award 2019-14)
    • Principle Award Recipient: BairdTimothy
  • Advance Queensland (Award AQRF13016-17RD2)
    • Principle Award Recipient: SarovichDerek S.
  • Advance Queensland (Award AQIRF0362018)
    • Principle Award Recipient: PriceErin P.
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001593
2022-10-27
2024-04-21
Loading full text...

Full text loading...

/deliver/fulltext/jmm/71/10/jmm001593.html?itemId=/content/journal/jmm/10.1099/jmm.0.001593&mimeType=html&fmt=ahah

References

  1. Breidenstein EBM, de la Fuente-Núñez C, Hancock REW. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol 2011; 19:419–426 [View Article]
    [Google Scholar]
  2. Botelho J, Grosso F, Peixe L. Antibiotic resistance in Pseudomonas aeruginosa - mechanisms, epidemiology and evolution. Drug Resist Updat 2019; 44:100640 [View Article]
    [Google Scholar]
  3. Schwartz T, Volkmann H, Kirchen S, Kohnen W, Schön-Hölz K et al. Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates. FEMS Microbiol Ecol 2006; 57:158–167 [View Article]
    [Google Scholar]
  4. Higgins PG, Fluit AC, Milatovic D, Verhoef J, Schmitz FJ. Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa. Int J Antimicrob Agents 2003; 21:409–413 [View Article]
    [Google Scholar]
  5. Poole K. Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2011; 2:65 [View Article]
    [Google Scholar]
  6. Lee JK, Lee YS, Park YK, Kim BS. Alterations in the GyrA and GyrB subunits of topoisomerase II and the ParC and ParE subunits of topoisomerase IV in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa. Int J Antimicrob Agents 2005; 25:290–295 [View Article]
    [Google Scholar]
  7. Yonezawa M, Takahata M, Matsubara N, Watanabe Y, Narita H. DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1995; 39:1970–1972 [View Article]
    [Google Scholar]
  8. Rehman A, Jeukens J, Levesque RC, Lamont IL. Gene-gene interactions dictate ciprofloxacin resistance in Pseudomonas aeruginosa and facilitate prediction of resistance phenotype from genome sequence data. Antimicrob Agents Chemother 2021; 65:e0269620 [View Article]
    [Google Scholar]
  9. Micek ST, Lloyd AE, Ritchie DJ, Reichley RM, Fraser VJ et al. Pseudomonas aeruginosa bloodstream infection: importance of appropriate initial antimicrobial treatment. Antimicrob Agents Chemother 2005; 49:1306–1311 [View Article]
    [Google Scholar]
  10. Darie AM, Khanna N, Jahn K, Osthoff M, Bassetti S et al. Fast multiplex bacterial PCR of bronchoalveolar lavage for antibiotic stewardship in hospitalised patients with pneumonia at risk of Gram-negative bacterial infection (Flagship II): a multicentre, randomised controlled trial. Lancet Respir Med 2022S2213-2600(22)00086-8 [View Article]
    [Google Scholar]
  11. Nguyen KV, Nguyen TV, Nguyen HTT, Le DV. Mutations in the gyrA, parC, and mexR genes provide functional insights into the fluoroquinolone-resistant Pseudomonas aeruginosa isolated in Vietnam. Infect Drug Resist 2018; 11:275–282 [View Article]
    [Google Scholar]
  12. Rehman A, Patrick WM, Lamont IL. Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: new approaches to an old problem. J Med Microbiol 2019; 68:1–10 [View Article]
    [Google Scholar]
  13. Kos VN, Déraspe M, McLaughlin RE, Whiteaker JD, Roy PH et al. The resistome of Pseudomonas aeruginosa in relationship to phenotypic susceptibility. Antimicrob Agents Chemother 2015; 59:427–436 [View Article]
    [Google Scholar]
  14. Birdsell DN, Pearson T, Price EP, Hornstra HM, Nera RD et al. Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models. PLoS One 2012; 7:e32866 [View Article]
    [Google Scholar]
  15. Germer S, Higuchi R. Single-tube genotyping without oligonucleotide probes. Genome Res 1999; 9:72–78 [View Article]
    [Google Scholar]
  16. Donà V, Smid JH, Kasraian S, Egli-Gany D, Dost F et al. Mismatch amplification mutation assay-based real-time PCR for rapid detection of Neisseria gonorrhoeae and antimicrobial resistance determinants in clinical specimens. J Clin Microbiol 2018; 56:e00365–00318 [View Article]
    [Google Scholar]
  17. Sarovich DS, Price EP, Von Schulze AT, Cook JM, Mayo M et al. Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia. PLoS One 2012; 7:e30789 [View Article]
    [Google Scholar]
  18. Germer S, Holland MJ, Higuchi R. High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR. Genome Res 2000; 10:258–266 [View Article]
    [Google Scholar]
  19. Webb KA, Olagoke O, Baird T, Neill J, Pham A et al. Genomic diversity and antimicrobial resistance of Prevotella species isolated from chronic lung disease airways. Microb Genom 2022; 8:000754 [View Article]
    [Google Scholar]
  20. McCarthy KL, Paterson DL. Long-term mortality following Pseudomonas aeruginosa bloodstream infection. J Hosp Infect 2017; 95:292–299 [View Article]
    [Google Scholar]
  21. Freschi L, Jeukens J, Kukavica-Ibrulj I, Boyle B, Dupont M-J et al. Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium. Front Microbiol 2015; 6:1036 [View Article]
    [Google Scholar]
  22. Anuj SN, Whiley DM, Kidd TJ, Bell SC, Wainwright CE et al. Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes. Diagn Microbiol Infect Dis 2009; 63:127–131 [View Article]
    [Google Scholar]
  23. Stewart AG, Price EP, Schabacker K, Birikmen M, Harris PNA et al. Molecular epidemiology of third-generation-cephalosporin-resistant Enterobacteriaceae in southeast Queensland, Australia. Antimicrob Agents Chemother 2021; 65:e00130–21 [View Article]
    [Google Scholar]
  24. Price EP, Viberg LT, Kidd TJ, Currie BJ et al. Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung. Microb Genom 2018; 4:e000194 [View Article]
    [Google Scholar]
  25. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article]
    [Google Scholar]
  26. Madden DE, Baird T, Bell SC, McCarthy KL. Keeping up with the pathogens: Improved antimicrobial resistance detection and prediction in Pseudomonas aeruginosa. MedRxiv 2022 [View Article]
    [Google Scholar]
  27. Madden DE, Webb JR, Steinig EJ, Currie BJ, Price EP et al. Taking the next-gen step: Comprehensive antimicrobial resistance detection from Burkholderia pseudomallei. EBioMedicine 2021; 63:103152 [View Article]
    [Google Scholar]
  28. de Lamballerie X, Zandotti C, Vignoli C, Bollet C, de Micco P. A one-step microbial DNA extraction method using “Chelex 100” suitable for gene amplification. Res Microbiol 1992; 143:785–790 [View Article]
    [Google Scholar]
  29. van Belkum A, Soriaga LB, LaFave MC, Akella S, Veyrieras J-B et al. Phylogenetic distribution of CRISPR-cas systems in antibiotic-resistant Pseudomonas aeruginosa. mBio 2015; 6:e01796–15 [View Article]
    [Google Scholar]
  30. Cabot G, López-Causapé C, Ocampo-Sosa AA, Sommer LM, Domínguez et al. Deciphering the resistome of the widespread Pseudomonas aeruginosa sequence type 175 international high-risk clone through whole-genome sequencing. Antimicrob Agents Chemother 2016; 60:7415–7423 [View Article]
    [Google Scholar]
  31. Del Barrio-Tofiño E, López-Causapé C, Cabot G, Rivera A, Benito N et al. Genomics and susceptibility profiles of extensively drug-resistant Pseudomonas aeruginosa isolates from Spain. Antimicrob Agents Chemother 2017; 61:1–13 [View Article]
    [Google Scholar]
  32. Sherrard LJ, Tai AS, Wee BA, Ramsay KA, Kidd TJ et al. Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis. PLoS One 2017; 12:1–15 [View Article]
    [Google Scholar]
  33. Fraser TA, Bell MG, Harris PNA, Bell SC, Bergh H et al. Quantitative real-time PCR assay for the rapid identification of the intrinsically multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia. Microb Genom 2019; 5:e000307 [View Article]
    [Google Scholar]
  34. Tsang KK, Maguire F, Zubyk HL, Chou S, Edalatmand A et al. Identifying novel β-lactamase substrate activity through in silico prediction of antimicrobial resistance. Microb Genom 2021; 7:000500 [View Article]
    [Google Scholar]
  35. Buhl M, Kästle C, Geyer A, Autenrieth IB, Peter S et al. Molecular evolution of extensively drug-resistant (XDR) Pseudomonas aeruginosa strains from patients and hospital environment in a prolonged outbreak. Front Microbiol 2019; 10:1742 [View Article]
    [Google Scholar]
  36. Ramanathan B, Jindal HM, Le CF, Gudimella R, Anwar A et al. Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa. PLoS One 2017; 12:e0182524 [View Article]
    [Google Scholar]
  37. Farahi RM, Ali AA, Gharavi S. Characterization of gyra and parc mutations in ciprofloxacin-resistant Pseudomonas aeruginosa isolates from tehran hospitals in iran. Iran J Microbiol 2018; 10:242–249
    [Google Scholar]
  38. Takenouchi T, Sakagawa E, Sugawara M. Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones. Antimicrob Agents Chemother 1999; 43:406–409 [View Article]
    [Google Scholar]
  39. Akasaka T, Tanaka M, Yamaguchi A, Sato K. Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob Agents Chemother 2001; 45:2263–2268 [View Article]
    [Google Scholar]
  40. Kidd TJ, Ritchie SR, Ramsay KA, Grimwood K, Bell SC et al. Pseudomonas aeruginosa exhibits frequent recombination, but only a limited association between genotype and ecological setting. PLoS One 2012; 7:e44199 [View Article]
    [Google Scholar]
  41. Bergkessel M, Guthrie C. Colony PCR. Methods Enzymol 2013; 529:299–309 [View Article]
    [Google Scholar]
  42. Levin BR, Perrot V, Walker N. Compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria. Genetics 2000; 154:985–997 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001593
Loading
/content/journal/jmm/10.1099/jmm.0.001593
Loading

Data & Media loading...

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