Aac(6 )-Ib-cr genotyping by simultaneous high resolution melting analysis of an unlabelled probe and full length amplicon

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1 AAC Accepts, published online ahead of print on 14 December 2009 Antimicrob. Agents Chemother. doi: /aac Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Aac(6 )-Ib-cr genotyping by simultaneous high resolution melting analysis of an unlabelled probe and full length amplicon Running title: Aac(6 )-Ib-cr genotyping by HRM Jan M Bell 1, John D Turnidge 1,2, Patiyan Andersson 1 * 1 SA Pathology, Women s and Children s Hospital, 72 King William Rd, North Adelaide, SA 5006, Australia. 2 Departments of Pathology, Paediatrics and Molecular and Biomedical Sciences, Adelaide University, Adelaide, SA 5000, Australia *Corresponding author. Department of Microbiology and Infectious Diseases, Women s and Children s Hospital, 72 King William Rd, North Adelaide, SA 5006, Australia. Tel: ; Fax: ; Patiyan.andersson@gmail.com Key words: aac(6 )-Ib-cr, high resolution melting, unlabelled probe, LC480 We have developed a time and cost efficient one-step closed-tube assay for genotyping of aac(6 )-Ib-cr, capable of distinguishing between the two genetic aac(6 )-Ib-cr variants. Genotyping uses the combined information of simultaneously acquired high resolution melting data from an unlabelled probe and the full length amplicon. There are several variants of the plasmid-borne gene encoding aminoglycoside acetyltransferase aac(6 )-Ib, and of greatest clinical concern is the aac(6 )-Ib-cr variant which extends the enzyme targets to include fluoroquinolones in addition to aminoglycosides (2, 4, 6, 7, 9). The aac(6 )-Ib-cr gene has spread rapidly among Enterobacteriaceae and although only conferring a low-level resistance, it may create an environment facilitating the selection of higher resistance determinants, especially those harbouring one topoisomerase mutation. The aac(6 )-Ib-cr variant is characterised by amino acid changes at codon 102 (Trp Arg) and codon 179 (Asp Tyr) (9). We have designed an assay which distinguishes between the three known alleles found in the aac(6 )-Ib gene at codon 102. There are two described aac(6 )-Ib-cr variants which differ from the wild type aac(6 )-Ib (accession no. AF322577) (12) [hereafter denoted wt] at nucleotide 304 by a T C (accession no. EF100892, EU675686) (1, 11) [here in after denoted cr-c] or T A (accession no. EF636461, EF443074, EU54272) (5, 8, 10) [here in after denoted cr-a] change. Both nucleotide changes result in a Trp Arg amino acid substitution. On a high-resolution melting capable real-time PCR platform SNP class 1 mutations (C/T or G/A) are easily detected due to the drastic change in the number of hydrogen bonds, which in short amplicons result in significant changes in melting characteristics. However, due to the subtle nature of SNP class 4 mutations (A/T) where no changes in number of hydrogen bonds occur, these are notoriously difficult to reliably detect and differentiate from the normal variation of the instrument. We have addressed this problem by combining information from two simultaneous melting events obtained in a single reaction. A high resolution melt of a short amplicon containing nucleotide 304, enables easy identification of the cr-c, which has a significantly higher melting temperature, but the cr-a cannot be distinguished from the wt. An unlabelled probe with a perfect match to the wt allele was included, which upon melting allowed discrimination between the wt and the two cr variants, where the latter two have lower melting temperatures due to the mismatch at the polymorphic site. The reaction was changed to contain asymmetric concentrations of primers in order to promote amplification of the DNA strand complimentary to the probe. The combined information from the two separate melting events results in an unambiguous genotype call.

2 The assay was developed using a set of 12 isolates, 3 isolates for each known aac(6 )-Ib genotype: wt, cr-c, cr-a and wt/cr-c (heterozygote). The genotype was determined by RFLP using BtsCI (New England Biolabs) (6) and confirmed by sequencing. Primers and probes were designed in Primer3 ( using AF as template. Primers were aacib_hrm_f 5 - AGTCGTACGTTGCTCTTGGA-3 and aacib_hrm_r 5 -CCTGGATCGGTTTCTTCTTC-3, which amplified a 58bp product. The probe, denoted aacib_hrm_probe 5 - AGCGGGGACGGATGGTGGGAAGAAG-3 (25bp), was phosphorylated in the 3 end to prevent extension. Amplification was carried out in a 10 l reaction consisting of 1X LC480 HRM Master Mix (Roche), 3mM MgCl 2 (Roche), 5% Q-solution (Qiagen), with primer and probe concentrations as described below. Through testing of different primer ( M and M respectively) and probe ( M) concentrations and degrees of primer asymmetry (1:5 and 1:10), it was determined that optimal visualisation of both melting events was achieved with 0.05 M aacib_hrm_f, 0.25 M aacib_hrm_r (1:5 ratio) and 0.5 M aacib_probe. A higher concentration of probe disrupted the amplification and masked the amplicon melting, while a 1:10 ratio of primers lead to poor amplification and low amplicon yield. Different dilutions of template DNA (1:50 and 1:100) were also tested, where a 1:100 dilution of the template DNA enhanced the reproducibility of the assay, by reducing the effect of varying salt concentrations between different DNA preparations. PCR and subsequent high resolution melt was carried out on a LightCycler 480, with the following temperature profile: 95 C for 10min, 45 cycles of [95 C for 10s, 57 C for 20s, 72 C for 20s], 95 C for 1min, 40 C for 1min, followed by high resolution melting from 50 C to 99 C, with 25 acquisitions/ C. The results were analysed in the Gene Scanning mode of the LightCycler 480 software package. Two melting events were observed, the first around C, describing the disassociation of the probe. The second, occurring around C, shows the melting of the whole amplicon. The derivate melting curves for each genotype are presented in Figure 1. In brief, the probe and amplicon melting points were; 72.0 C and 81.9 C for wt, 69.7 C and 82.7 C for cr-c and 68.8 C and 81.7 C for cr-a. Heterozygote isolates containing both wt and cr alleles showed a distinct shoulder on the amplicon melting peak, which can be attributed to the melting of heteroduplex fragments. In the case of wt/cr-a heterozygote the probe melting event showed two distinct peaks representative of the two individual allele peaks, and the wt/cr-c heterozygote showed a flat-topped peak stretching across the individual allele peaks. All genotypes were easily identified and could be unambiguously called. This assay was also shown to perform successfully on the Corbett 6000 real-time platform (Qiagen). The assay was validated on a cohort of isolates (n=211), composed of Klebsiella pneumoniae (n=92), Escherichia coli (n=59), Enterobacter cloacae (n=35), Serratia marcescens (n=12), Proteus mirabilis (n=7), Citrobacter freundii (n=4), Enterobacter aerogenes (n=1), Providencia stuartii (n=1), for which the aac(6 )- Ib status (91 positive and 120 negative) and genotype as determined by RFLP was known (32 wt, 37 cr, 22 wt/cr), but to which the operator was blinded. Seven samples previously negative in the RFLP assay, were positive in the HRM probe assay. This was determined by sequencing to be due to a partial deletion of the gene destroying one of the primer binding sites in the RFLP assay. The results for the remaining 204 isolates were 100% concordant with the previously determined genotype. The assay was subsequently used to test a cohort of isolates with unknown aac(6 )-Ib genotype (n=732), composed of Enterobacter cloacae (n=308), Klebsiella pneumoniae (n=168), Escherichia coli (n=144), Enterobacter aerogenes (n=53), Klebsiella oxytoca (n=38), Enterobacter sakazakii (n=4), Enterobacter hormaechei (n=3), Enterobacter asburiae (n=2), Enterobacter amnigenus (n=1), Enterobacter gergoviae (n=1), Enterobacter spp. (n=1), Acinetobacter baumannii (n=4), Acinetobacter haemolyticus (n=1). The genotype distribution in this cohort was 84 wt (11.5%), 84 cr-c (11.5%), 10 cr-a (1.4%), 10 wt/cr-c (1.4%), 7 wt/cr-a (1.0%). The remaining 537 were negative for the aac(6 )-Ib gene. Sequencing traces of heterozygote isolates (wt/cr-c and wt/cr-a) showed double peaks of equal amplitude at nucleotide 304, indicating that the two alleles are present in equal quantities in the isolate. One strain showed peaks similar to that of a wt but with 0.5 o C higher melting points. Five isolates displayed amplicon peaks with shoulders indicative of heterozygotes, but with uneven probe

3 melting peaks. We believe this can be explained by an asymmetric ratio of the two alleles. Among our isolates coexistence of wt and cr alleles in an isolate was a surprisingly common phenomenon, an observation which was very recently supported in a French study (3). We hypothesise that these represent isolates where a proportion of the cells have acquired a cr allele, and under sustained antibiotic pressure these cells would quickly dominate the population. We have developed a one-step closed-tube assay for the detection and genotyping of aac(6 )-Ib-cr, capable of differentiating between the two genetic variants responsible for the aac(6 )-Ib-cr phenotype, which from an epidemiological perspective is of importance. The assay is rapid at <2 hours and costs less than US $1 per isolate. This assay addresses the need of an efficient detection system for the monitoring of the growing prevalence of clinically significant aac-ib-cr variants.

4 References 1. Ambrozic Avgustin, J., R. Keber, K. Zerjavic, T. Orazem, and M. Grabnar Emergence of the quinolone resistance-mediating gene aac(6')-ib-cr in extended-spectrum-beta-lactamaseproducing Klebsiella isolates collected in Slovenia between 2000 and Antimicrob Agents Chemother 51: Corvec, S., L. Cremet, N. Caroff, S. Dauvergne, R. Le Floch, A. Reynaud, D. Lepelletier, and P. Bemer Klebsiella pneumoniae clinical isolate coproducing SHV-2a, DHA-1, QnrB4, and AAC(6')-Ib-cr determinants in France. Diagn Microbiol Infect Dis 64: Guillard, T., V. Duval, H. Moret, L. Brasme, V. Vernet-Garnier, and C. de Champs Rapid detection of quinolone resistance gene aac(6')-ib-cr by pyrosequencing. J Clin Microbiol. 4. Kim, E. S., J. Y. Jeong, J. B. Jun, S. H. Choi, S. O. Lee, M. N. Kim, J. H. Woo, and Y. S. Kim Prevalence of aac(6')-ib-cr encoding a ciprofloxacin-modifying enzyme among Enterobacteriaceae blood isolates in Korea. Antimicrob Agents Chemother 53: Ma, J., Z. Zeng, Z. Chen, X. Xu, X. Wang, Y. Deng, D. Lu, L. Huang, Y. Zhang, J. Liu, and M. Wang High prevalence of plasmid-mediated quinolone resistance determinants qnr, aac(6')- Ib-cr, and qepa among ceftiofur-resistant Enterobacteriaceae isolates from companion and foodproducing animals. Antimicrob Agents Chemother 53: Park, C. H., A. Robicsek, G. A. Jacoby, D. Sahm, and D. C. Hooper Prevalence in the United States of aac(6')-ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother 50: Park, Y. J., J. K. Yu, S. Lee, E. J. Oh, and G. J. Woo Prevalence and diversity of qnr alleles in AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Serratia marcescens: a multicentre study from Korea. J Antimicrob Chemother 60: Quiroga, M. P., P. Andres, A. Petroni, A. J. Soler Bistue, L. Guerriero, L. J. Vargas, A. Zorreguieta, M. Tokumoto, C. Quiroga, M. E. Tolmasky, M. Galas, and D. Centron Complex class 1 integrons with diverse variable regions, including aac(6')-ib-cr, and a novel allele, qnrb10, associated with ISCR1 in clinical enterobacterial isolates from Argentina. Antimicrob Agents Chemother 51: Robicsek, A., J. Strahilevitz, G. A. Jacoby, M. Macielag, D. Abbanat, C. H. Park, K. Bush, and D. C. Hooper Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 12: Shi, W., J. Qin, and Z. Mi A Klebsiella pneumoniae sputum culture isolate from China carrying blaoxa-1, blactx-m-55 and aac(6')-ib-cr. J Med Microbiol 57: Wei, Q., X. Jiang, Z. Yang, N. Chen, X. Chen, G. Li, and Y. Lu dfra27, a new integronassociated trimethoprim resistance gene from Escherichia coli. J Antimicrob Chemother 63: Yan, J. J., W. C. Ko, and J. J. Wu Identification of a plasmid encoding SHV-12, TEM-1, and a variant of IMP-2 metallo-beta-lactamase, IMP-8, from a clinical isolate of Klebsiella pneumoniae. Antimicrob Agents Chemother 45:

5 30 -df/dt Fluorescence -df/dt Fluorescence -df/dt Fluorescence A B C Temperature wt cr-c cr-a Temperature wt cr-c wt/cr-c Temperature wt cr-a wt/cr-a

6 FIGURE LEGEND Figure 1. Derivate melting curves showing known genotypes of aac(6 )-Ib. Two melting events can be observed, the first at 65-75ºC describing the probe disassociation and the second at 79-84ºC describing the full length amplicon melting. A. Genotypes wt, cr-c and cr-a. The three genotypes can be distinguished in the probe melting event, but while the amplicon melting peak of the of a cr-c can be distinguished, the wt and cr-a are virtually identical. B. Wt and cr-c alleles coexisting in an isolate, creating a wt/cr-c heterozygous genotype. These heterozygotes are identified by a shoulder on the amplicon melting peak and a probe peak spanning the wt and cr-c probe peaks. Melting curves for the individual wt and cr-c genotypes are included in grey. C. Wt and cr-a alleles coexisting in an isolate, creating a wt/cr-a heterozygous genotype. These heterozygotes are identified by a shoulder on the amplicon melting peak and dual probe peaks representative of the wt and cr-a probe peaks. Melting curves for the individual wt and cr-a genotypes are included in grey. Downloaded from on January 14, 2019 by guest