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Antimicrob Agents Chemother. May 2007; 51(5): 1865–1868.
Published online Feb 16, 2007. doi:  10.1128/AAC.01341-06
PMCID: PMC1855555

Tetracycline Resistance in Group A Streptococci: Emergence on a Global Scale and Influence on Multiple-Drug Resistance[down-pointing small open triangle]

Abstract

A global sample of group A streptococci (GAS) revealed ≥80 separate acquisitions of tetracycline resistance. Of 244 clones, 38 and 25% displayed resistance to tetracycline and erythromycin, respectively; a relatively high proportion (15%) were resistant to both classes of drugs. tet(M) displayed a highly significant association with erm(B).

Tetracycline in the environment can shape the evolution of many bacteria. In gram positive organisms, resistance to tetracycline is typically conferred by ribosome protection genes, such as tet(M) and tet(O). The number of acquisitions of resistance to tetracycline among group A streptococci (GAS) is unknown. Using a global collection of GAS isolates of defined genetic backgrounds, the number of independent acquisitions of tetracycline resistance is estimated. Since tetracycline resistance genes can reside on mobile genetic elements that carry macrolide resistance genes (2, 4, 8), the co-occurrence of resistance to both classes of drugs was also assessed.

emm types and multilocus sequence types (STs), which are based on seven housekeeping loci, were previously reported for most GAS isolates analyzed (5, 7). Twenty-eight new ST profiles were identified by previously described methods (5).

Susceptibility versus resistance to erythromycin and tetracycline was established by the Etest (7) for 188 GAS isolates recovered from >20 countries; antibiotic resistance profiles were unknown at the onset of the study. Antibiotic susceptibility was defined by MICs of <0.25 and 2 μg/ml for erythromycin and tetracycline, respectively. All isolates were distinct in emm type and ST. Only 2.1% of the isolates were resistant to erythromycin, whereas 60 (31.9%) were resistant to tetracycline. Tetracycline-resistant GAS (TRGAS) outnumbered macrolide-resistant GAS (MRGAS) 15-fold.

Tetracycline resistance is usually acquired by GAS via horizontal gene transfer. The number of distinct acquisitions can be estimated by identifying distant genetic backgrounds associated with resistance. A set of 291 isolates, which includes ~79% of known emm types (Streptococcus pyogenes emm sequence database, available at http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm) and additional MRGAS (7), was screened for tetracycline resistance by the Etest and/or by PCR targeting tet(M) and tet(O) at an annealing temperature of 50°C using primers tetMF1-tetMR1 and tetOF1-tetOR1 (Table (Table1).1). TRGAS isolates (n = 112), represented by 90 STs from 27 countries (see Table S1 in the supplemental material), were identified.

TABLE 1.
Oligonucleotide primers used for PCR and nucleotide sequence determination of amplicons

Genetic distances between the TRGAS isolates were defined by the eBURST clustering algorithm for analyzing relationships between STs (3). STs sharing five or six housekeeping alleles—double- or single-locus variants, respectively—were assigned to the same clonal complex (CC). Among the 90 STs, 61 were singletons that differed from all other STs by ≥3 housekeeping alleles (Table (Table2);2); each singleton ST represents an independent acquisition of tetracycline resistance. Two emm12 ST36 isolates had distinct tet genes—tet(M) and tet(O)—bringing the subtotal to 62 acquisitions.

TABLE 2.
Conservative estimate for the number of independent acquisitions of tetracycline resistance among TRGAS isolates

Thirteen CCs account for the remaining 29 STs (Table (Table2).2). If each CC arose from a single TRGAS progenitor, then acquisition of resistance by organisms belonging to the same CC should be scored as a single genetic event. Accordingly, ≥75 independent acquisitions of tetracycline resistance are estimated. Closer inspection of the 13 CCs reveals five sets of double-locus variants in which STs of each pair also differ in the emm type, signifying that ≥3 sequential genetic steps were required to evolve these CCs. If differences in two housekeeping alleles, plus a different emm type, represent at least two independent acquisitions of resistance, then the minimum estimate for separate acquisitions of tetracycline resistance among GAS rises to ≥80 events.

For representative TRGAS isolates corresponding to the 80 independent acquisitions, the vast majority (n = 72; 90%) have tet(M). Distinctions between the tet(M) and tet(O) amplicons were confirmed by nucleotide sequencing using the primers listed in Table Table1.1. The findings for 49 TRGAS are summarized in a phylogenetic tree (Fig. (Fig.1).1). Two major sequence clusters distinguish the tet(M) and tet(O) alleles. The high levels of sequence similarity observed with tet(M) and tet(O) genes from other bacterial species provide additional support for the acquisition of tetracycline resistance genes by GAS via horizontal transfer.

FIG. 1.
Phylogenetic tree of partial tet(M) and tet(O) gene sequences. Partial tet(M) and tet(O) nucleotide sequences are shown for 49 TRGAS isolates (GenBank accession numbers ...

In streptococci, mobile genetic elements carrying tet(M) or tet(O) sometimes harbor genes encoding macrolide resistance (1, 4). Since selection for resistance to one antibiotic can influence the evolution of resistance to another drug in multiply resistant bacteria, it was of interest to determine the relationship between resistance to tetracycline and macrolides in GAS. The resistance genotype of MRGAS was determined by PCR for erm(A), erm(B), and mef(A) and has been reported previously for most isolates (7). The degree of growth inhibition by erythromycin was determined for other isolates.

Findings on susceptible phenotypes and resistant genotypes were combined with data on the emm type and ST. A set of 244 distinct GAS isolates was identified in which each isolate had a unique profile comprising the erythromycin susceptibility or macrolide resistance genotype, tetracycline susceptibility or tetracycline resistance genotype, and emm type-ST combination. Furthermore, all 244 isolates sharing the same antibiotic resistance profile were distant by ≥2 genetic steps; they are represented by 182 singleton STs and 21 CCs. Of the 244 distant clones, 92 (38%) were resistant to tetracycline and 60 (25%) were resistant to macrolides (Table (Table3).3). Thirty-six (15%) displayed resistance to both classes of drugs; 128 were susceptible to both. According to a two-by-two test for independence, the number of organisms resistant to both antibiotics exceeded the number of organisms expected to be doubly resistant simply by chance (P < 0.001 by a two-tailed Fisher exact test). Findings were similar for the 239 isolates differing by ≥3 genetic steps.

TABLE 3.
Distribution of resistance genes among 244 genetically distinct GAS isolatesa

Of the 92 TRGAS clones, 81 (88%) harbor tet(M) (Table (Table3).3). The tet(M) gene is found in association with erm(B) in 12 isolates, representing 71% of the total erm(B)-positive isolates. A two-by-two test for independence was used to compare the number of isolates observed to have both tet(M) and erm(B) (n = 12), tet(M) only (n = 69), erm(B) only (n = 5), or neither (n = 158), to the number of isolates expected if tet(M) and erm(B) were randomly associated. The frequency of co-occurrence of tet(M) and erm(B) is significantly greater than that expected by chance (P = 0.002). In contrast, the erm(A) gene co-occurs with tet(M) at a frequency that can be explained by chance, as does mef(A) (P, not significant).

The data support the hypothesis that tet(M) and erm(B) are often linked, and they are consistent with the idea that tet(M) and erm(B) may be coinherited. In streptococci, tet(M) is often carried by the conjugative transposon Tn916 or the related Tn1545, which together have a broad bacterial host cell range (2, 6). Although mobile genetic elements carrying tet(M) were not identified in the TRGAS, members of the Tn916-Tn1545 family of elements are strong candidates. In addition to tet(M), the Tn1545 element harbors erm(B) (8). Since tet(M) is more widely distributed than erm(B), and >70% of erm(B) occurrences are found in tet(M)-positive GAS, it seems plausible that strong selection forces act to drive the spread of tet(M) among many different GAS strains, whereby erm(B) is the frequent hitchhiker.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Jeanette Sutherland, Stephen Henry, Zerina Kratovac, and Nutan Bhaskar for technical assistance and Adrian Whatmore and Karen McGregor for early release of MLST data.

This work was supported by NIH grants AI061454, AI065572, and GM060793.

Footnotes

[down-pointing small open triangle]Published ahead of print on 16 February 2007.

Supplemental material for this article may be found at http://aac.asm.org/.

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