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Pyrosequencing was used to rapidly detect aac(6′)-Ib and aac(6′)-Ib-cr genes. This plasmid-mediated quinolone resistance determinant is increasing in extended-spectrum beta-lactamase-producing Enterobacteriaceae. This method is faster and more cost-effective than the methods previously described. Sequences obtained with this pyrosequencing method showed 100% concordance with conventional sequencing.
Quinolone resistance in Enterobacteriaceae usually results from mutations in genes carried by chromosomally encoded type II topoisomerases, efflux pumps, or porin-related proteins. Recently, plasmid-mediated quinolone resistances (PMQR) have been described. They have been reported worldwide in unrelated enterobacterial species and are usually associated with mobile elements (17). Quinolones and beta-lactams are among the three most commonly used antimicrobials in human therapeutics. PMQR determinants confer low-level resistance, but their presence could potentially facilitate evolution of the bacterial host toward higher levels of resistance by mutational alterations in type II topoisomerases. PMQR determinants are often combined with extended-spectrum beta-lactamases (ESBLs). This suggests that there is the potential for selection of PMQR by the use of beta-lactams and of beta-lactam resistance by the use of quinolones. The Qnr proteins protect DNA from quinolone binding to gyrase and topoisomerase IV (10, 22). To date, 6 qnrA, 20 qnrB, 1 qnrC, 1 qnrD, and 4 qnrS genes have been identified (3, 9, 23). QepA1 and QepA2 are quinolone efflux pump proteins (2, 14). AAC(6′)-Ib-cr is a variant of AAC(6′)-Ib and is responsible for reduced susceptibility to ciprofloxacin or norfloxacin by N acetylation of a piperazinyl amine. AAC(6′)-Ib-cr differs from AAC(6′)-Ib by the following two amino acid substitutions: Trp102Arg and Asp179Tyr (18). Sequence alignments of the genes confirm that Trp102Arg and Asp179Tyr are due to single nucleotide polymorphisms (SNPs) T304C/A and G535C, respectively (Fig. (Fig.11).
Pyrosequencing is suitable for mutation analysis (4, 7, 8) and has already been used for detecting resistance-conferring genes (11, 12, 16, 21). To our knowledge, this is the first study to describe the use of pyrosequencing as a tool for the rapid detection of aac(6′)-Ib-cr and aac(6′)-Ib genes.
The method was first used for the rapid detection of aac(6′)-Ib-cr in 135 ESBL-producing Enterobacteriaceae isolates collected in 2004 in the Champagne-Ardenne region in France. Enterobacter cloacae HM 477 (kindly provided by Emmanuelle Cambau) was used as an aac(6′)-Ib positive control.
DNA extraction was performed with the QIAamp DNA minikit (Qiagen, Courtaboeuf, France). Two PCR assays were realized with the MyCycler thermocycler (Bio-Rad, Marnes-la-Coquette, France). One PCR assay targeted the 77-base-pair (bp) portion with T304C/A, and another one targeted the 102-bp portion with G535T. The forward primers were covalently coupled to biotin at the 5′ end to obtain a pyrosequencing template from the PCR product. A 77-bp amplicon and a 102-bp amplicon were generated in a 50-μl PCR that contained 5 μl of target DNA, 5 μl of PCR buffer, 0.5 μl of MgCl2 (25 mM), 0.5 μl of deoxynucleoside triphosphate (dNTP) (25 mM), 2 μl of T304C-Fbiotin or G535T-Fbiotin (25 μM), 2 μl of T304C-R or G535T-R (25 μM) (Table (Table1),1), 0.5 μl of Taq (5 U/μl), and 34.5 μl of sterile water. The PCR consisted of the following: denaturation and enzyme activation at 95°C for 3 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and elongation at 72°C for 30 s, followed by a final extension at 72°C for 5 min. PCR products of the expected size were verified by electrophoresis in a 2% agarose gel.
The crude biotinylated single-stranded DNA was converted to pure single-stranded DNA templates using Streptavidin Sepharose HP (GE Healthcare, Pittsburgh, PA). Strand elution and subsequent washing were realized by vacuum filtration using the vacuum prep tool (Biotage, Uppsala, Sweden), in accordance with the manufacturer's instructions. The nucleotides were sequentially dispensed according to the sequence data loaded, as follows: C(A/G/T)TCCGTCCC for T304C/A and CAT(A/C)TGGGG for G535T. Light was produced whenever a nucleotide formed a base pair with the next available complementary base in the DNA template strand. Light signals were detected on a pyrogram, on which each peak corresponds to one or more identical bases added to the growing DNA strand. The pyrograms were analyzed using standard PSQ 96MA system software version 2.01 (Biotage, Uppsala, Sweden).
All sequences obtained by pyrosequencing were verified by sequencing (20) the PCR products that were amplified with T304C-F and G535T-R from DNA templates of strains carrying aac(6′)-Ib-cr and strains carrying aac(6′)-Ib.
All of the 135 tested isolates rendered interpretable sequences. All of the results are listed in Table Table2.2. T304 and G535 were detected on E. cloacae HM 477 at each run as positive controls. Incorporation of A [complementary to T304 for aac(6′)-Ib], T, or G [complementary to A304 or C304 for aac(6′)-Ib-cr] and incorporation of C [complementary to G535 for aac(6′)-Ib] or A [complementary to T535 for aac(6′)-Ib-cr] were easily detected on pyrograms (Fig. (Fig.2).2). Sequences were in complete agreement with conventional sequencing. None of the aac(6′)-Ib-cr genes detected in this study carried the SNP T304A. None of the isolates carrying aac(6′)-Ib-cr or heterozygous isolates were positive for qnr genes (6). All of the isolates positive for aac(6′)-Ib-cr also carry blaCTX-M-15 (1), which is described in the literature (5, 15).
The pyrosequencing method described in this paper is a rapid tool for the detection of aac(6′)-Ib-cr and aac(6′)-Ib. From extraction to sequencing, the cost for one test is approximately $3, compared to $10 for previously described methods using PCR restriction, followed by conventional sequencing (5, 13). The manual setup of each experiment took less than 4 h (for 46 isolates), compared to 2 days for previously described methods. It is faster and more cost-effective than the methods previously described, which have the double disadvantage of being both labor-intensive and time-consuming. The pyrosequencing method described here is convenient for preventing usage of inefficient quinolone treatments, leading to potential therapeutic failures. Indeed, the presence of aac(6′)-Ib-cr facilitates the selection of chromosomal mutants in the presence of a quinolone (18), and in specific geographical areas where aac(6′)-Ib-cr prevalence is high (13, 18, 19), this method could be used to detect this enzyme rapidly. In addition, the pyrosequencing technique may provide a rapid tool for the surveillance of antibiotic resistance determinants in epidemiological studies.
We thank Lydie Morcrette from PRBI for her excellent technical assistance, Emmanuelle Cambau (Hôpital Saint Louis, AP-HP, France) for kindly providing strain HM 477, and Tim Greacen for the English revision.
Published ahead of print on 18 November 2009.