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Antimicrob Agents Chemother. Nov 2005; 49(11): 4798–4800.
PMCID: PMC1280148

Multiplex PCR for Simultaneous Detection of Macrolide and Tetracycline Resistance Determinants in Streptococci

Abstract

Resistance to macrolides and tetracyclines is increasing among streptococci and co-occurs as their resistance determinants are carried on the same mobile element. We developed a multiplex PCR to facilitate simultaneous and specific detection of resistance determinants for both macrolides [erm(A), erm(B), and mef(A/E)] and tetracyclines [tet(M), tet(O), tet(K), and tet(L)] in streptococci.

Macrolides and tetracyclines are among the most common antibiotics employed for the treatment of streptococcal infections. However, a concomitant increase in resistance to both these antibiotics has been observed among pathogenic and commensal streptococci, mostly because their major resistance determinants are carried on the same mobile element (2, 4, 9, 12, 13).

Resistance to macrolides in streptococci occurs by two main mechanisms. First, erm(B)/erm(A) gene products methylate specific residues in 23S rRNA and disable macrolide binding to its target (7). Rarely, mutations in 23S rRNA or in the ribosomal proteins L4 and L22 also modify the macrolide binding site, causing macrolide resistance (3, 16). The second major mechanism is active macrolide efflux, mediated by an ABC transporter. The transmembrane domains of this pump are encoded by mef genes [mef(A) in Streptococcus pyogenes and mef(E) in S. pneumoniae] and the ATP-binding domains by the msr(D) gene (F. Ianelli, M. Santagati, J.-D. Docquier, M. Cassone, M. R. Oggioni, G. Rossolini, S. Stefani, and G. Pozzi, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1188, 2004). Tetracycline resistance in streptococci is mediated by ribosomal protection proteins encoded mainly by the tet(M) or tet(O) genes. These proteins are homologous to elongation factors EF-G and EF-Tu and possess GTPase activity that is important in the displacement of tetracycline from the ribosome (1). In contrast to macrolide resistance, efflux pumps for tetracycline encoded by the tet(K) or tet(L) genes in streptococci are relatively uncommon (13).

PCR has been widely used to detect the presence of resistance genes in human or animal streptococci. However, because streptococci carrying more than one macrolide/tetracycline resistance determinant are increasingly being noted, many separate PCRs have to be performed to detect their presence (4, 9, 12, 13). Although the correlation between the presence of erm(B) and tet(M) is well established (2, 9) and there is also evidence suggesting a genetic linkage of tet(O) with erm(A) or mef(A) (4), assays for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci have not been previously developed. Here we describe a multiplex PCR procedure that enables simultaneous detection of three macrolide [erm(A), erm(B), and mef(A/E)] and four tetracycline [tet(M), tet(O), tet(K), and tet(L)] resistance determinants in streptococci.

(A preliminary account of this work was presented at the 44th International Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 30 October to 2 November 2004.)

We utilized the following resistant reference strains as positive controls: erm(A), S. pyogenes UR1092 (10); erm(B), S. pyogenes BM137 (9); mef(A) S. pyogenes STP046 (10); tet(M), S. pyogenes BM137 (9); tet(O), Enterococcus faecalis BM4110 (9); tet(K), S. aureus R-16794 (6) (BCCM/LMG Bacteria Collection, Ghent University, Belgium; http://bccm .belspo.be/db/bacteria_search.htm); and tet(L), E. faecalis P33 (BCCM/LMG Bacteria Collection). Genomic DNA was extracted either with GenElute Bacterial Genomic Miniprep (Sigma-Aldrich) or by alkaline lysis (14). For the latter, two to four bacterial colonies were emulsified in 20 μl lysis buffer (0.25% sodium dodecyl sulfate, 0.05 N NaOH) at 94°C for 5min. The lysates were diluted with 180 μl water and centrifuged at 16,000 × g for 5 min, and the supernatants were directly used as template DNA. The genotypes of the reference strains were confirmed with individual (singlex) PCRs using known primers for erm(A) (10), erm(B) (15), mef(A)/mef(E) (15), tet(M) (17), tet(O) (11), tet(K) (11), and tet(L) (11). All strains gave expected PCR products. Genomic DNA from the six reference strains was further pooled in equimolar amounts and served as a multiplex control for seven resistance determinants and one housekeeping control (see below). According to published guidelines (5), PCR conditions were optimized and the following considerations were taken while designing primers. One, cross-annealing andspecificity of the primers were examined using BLAST (www.ncbi.nlm.nih.gov). Two, primer pairs generating amplicons with at least a 50-bp difference were chosen to facilitate their electrophoretic separation. Three, the melting temperature was kept close to 60°C for all primer pairs. Thus, new primers for erm(B), mef, tet(K), and tet(L) genes were designed using Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and synthesized (Eurogentec, Seraing, Belgium) (Table (Table1).1). Four, consensus primers for mef were chosen that amplified both mef(A) and mef(E). Finally, consensus primers amplifying the 16S rRNA gene were designed that served as internal controls for all streptococci analyzed here (Table (Table11).

TABLE 1.
Primers used in the multiplex PCR assay

PCR was performed in a final volume of 50 μl of 0.8× PCR buffer (50 mM KCl, 10 mM Tris-HCl [pH 9.0], 0.1% Triton X-100, 0.01% [wt/vol] stabilizer, 1.5 mM MgCl2) containing 300 μM deoxynucleoside triphosphates, 3.5 mM MgCl2, 2 U Taq polymerase (Invitrogen, Carlsbad, Calif.), and 10 to 15 ng of template DNA. Optimized primer concentrations were as follows: erm(A), 0.5 μM; erm(B), 0.5 μM; mef(A/E), 0.2 μM; tet(M), 0.4 μM; tet(O), 0.3 μM; tet(L), 0.4 μM; tet(K), 0.1 μM; and 16S rRNA, 0.16 μM. PCR was performed on a DNA thermal cycler (9600 GeneAmp PCR System; Perkin-Elmer, Zaventem, Belgium) with the following cycling conditions: an initial cycle of 3 min at 93°C, 30 cycles of 1 min of denaturation at 93°C, 1 min of annealing at 62°C, and 4 min of extension at 65°C, followed by one cycle of 3 min of elongation at 65°C. PCR products were analyzed by electrophoresis in a 1.5% agarose gel at 150 V for 1.05 h in 0.5× TBE (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA) containing 0.05 mg/liter ethidium bromide. Visualization and image acquisition was performed with Gel-Doc-1000 (Bio-Rad Laboratories, Nazareth, Belgium). Using these conditions, the pooled positive control yielded eight expected bands (Fig. (Fig.1,1, lane C).

FIG. 1.
Agarose gel electrophoresis of multiplex PCR products as well their phenotypic description and resistance gene content. The control lane (C) amplifies all genes from DNA samples pooled from the six reference strains. Lane M, molecular size standard (100-bp ...

This technique was further validated on 125 previously characterized macrolide- and tetracycline-resistant (n = 95) and -sus ceptible (n = 30) streptococci, comprising S. pneumoniae (n = 42) (J. Van Eldere et al., unpublished data), S. pyogenes (n = 49) (8), and viridans streptococci (S. mitis [n = 14], S. parasanguinis [n = 5], S. thermophilus [n = 5], S. salivarius [n= 6], S. constellatus [n = 2], S. anginosus [n = 1], and S.cristatus [n = 1]) (9). Seven singlex PCRs and the multiplex PCR were performed on DNA extracted by two methods for these 125 isolates. The pooled DNA from the six reference strains served as a positive control ladder (Fig. (Fig.1).1). No discordance was noted between the two types of DNA extraction procedures for either singlex or multiplex PCR. Compared to singlex PCR results, the multiplex PCR identified genes with 100% sensitivity and specificity (Table (Table2).2). Thus, the described multiplex assay could be a useful tool to analyze seven commonly occurring antibiotic resistance genes in streptococci. In addition, the use of 16S rRNA as a housekeeping positive control would facilitate DNA and PCR quality control. Lastly, the multiplex PCR described here is not free from the limitations inherent to any PCR assay, i.e., false-negative data due to mutations in the primer-annealing region of the gene amplified or false-positive results caused by gene inactivation due to insertions and/or deletions in regions outside the PCR product. Thus, to correctly interpret PCR results, it is imperative to characterize resistance gene expression in bacterial isolates by means of phenotypic tests like MICs and disk diffusion. Notwithstanding these limitations, the single-step multiplex assay described here will prove economical in terms of labor, time, and cost for studies investigating large numbers of streptococci for macrolide and tetracycline resistance mechanisms.

TABLE 2.
Streptococcal isolates (n = 125) grouped according to their macrolide and tetracycline resistance gene content, as detected by the multiplex assay, and corresponding MIC rangesa

Acknowledgments

We are grateful to J. Van Eldere and J. Verhaegen for providing S. pneumoniae isolates and to G. Huys for the reference strains S. aureus R-16794 and E. faecalis P33.

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