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Antimicrob Agents Chemother. Aug 2007; 51(8): 3042–3043.
Published online Jun 11, 2007. doi:  10.1128/AAC.00249-07
PMCID: PMC1932502

SmaI Typeability and Tetracycline Susceptibility and Resistance in Streptococcus pyogenes Isolates with Efflux-Mediated Erythromycin Resistance[down-pointing small open triangle]

In Streptococcus pyogenes, efflux-mediated macrolide resistance is encoded by the mef(A) gene and is associated with a particular resistance pattern (M phenotype) characterized by resistance, usually at a low level, only to 14- and 15-membered macrolides (10). The mef(A) gene is carried by different genetic elements, depending on whether the isolates are susceptible or resistant to tetracycline (2). In the tetracycline-susceptible isolates, mef(A) is contained in a Tn1207.1 transposon, identical to the one originally described in S. pneumoniae (9), inserted into larger genetic elements of phage origin such as Tn1207.3 (8) or Φ10394.4, a related 58.8-kb chimeric element (1). In the tetracycline-resistant isolates, mef(A) is linked to the tet(O) gene in a tet(O)-mef(A) chimeric element which, unlike the Tn1207.3-Φ10394.4 family, may contain different, defective variants of the Tn1207.1 transposon and is not integrated into the chromosome within the comEC gene (2).

The most common method used to type macrolide-resistant streptococci involves restriction of genomic DNA with the SmaI endonuclease, followed by pulsed-field gel electrophoresis analysis. However, several studies have reported that some isolates with the M phenotype are nontypeable because their DNA is refractory to SmaI digestion. Since early studies, nontypeability was seen to be a characteristic of tetracycline-susceptible M-phenotype isolates (7, 11), typically carrying Tn1207.3 or Φ10394.4 (2, 6), whereas tetracycline-resistant M-phenotype isolates, typically carrying the tet(O)-mef(A) element (2), were usually restricted by SmaI (6). Subsequently, a DNA-modifying methyltransferase encoded by the Tn1207.3-Φ10394.4 family was described, which acts on the SmaI recognition sequence and renders DNA refractory to cleavage by SmaI (4). Further insights were recently gained by Euler et al. (3), who identified a type II restriction-modification (R-M) cassette on Φ10394.4—located just downstream of Tn1207.1 in the conserved region of phage origin—formed by three open reading frames (ORFs): two encoding two subunits of a restriction endonuclease and one (spyIM) encoding the methyltransferase responsible for DNA resistance to SmaI digestion.

Three different strains from our collection of M-phenotype S. pyogenes strains were investigated: m46 [SmaI typeable, tetracycline resistant, carrying the tet(O)-mef(A) element] (2, 5, 6, 7), and Spy-1 and Spy-2 (both SmaI nontypeable and tetracycline susceptible, carrying Tn1207.3 and Φ10394.4, respectively) (6, 7). PCR analysis revealed the R-M cassette in strains Spy-1 and Spy-2 but not in strain m46 (nor in other tetracycline-resistant isolates containing different defective variants of the Tn1207.1 transposon [6; data not shown]). The entire region was explored by pairing primers ORF8-for (ACCGATTTGCCTCACTGCAC) and dsPOL-rev (ACCATTAGAGATGACATTCG), targeting umuC-mucB (the last ORF of Tn1207.1) and the intergenic region immediately downstream of the phage DNA polymerase gene, respectively. Whereas an amplicon of the size (10,756 bp) expected on the basis of the published sequences of Tn1207.3 (GenBank accession no. AY657002) and Φ10394.4 (GenBank accession no. AY445042) was obtained from strains Spy-1 and Spy-2, a smaller amplicon (7,380 bp) was obtained from strain m46 (Fig. (Fig.1).1). The 7,380-bp amplicon was sequenced (EMBL accession no. AM492531); sequence analysis showed that the entire R-M cassette and the adjacent ORF of Tn1207.3-Φ10394.4 were replaced by three new ORFs encoding three hypothetical proteins. Two areas of homology with Tn1207.3-Φ10394.4 were detected, upstream (93% homology, including umuC-mucB) and downstream (94% homology, including the phage DNA polymerase gene) of the three new ORFs (Fig. (Fig.11).

FIG. 1.
ORF map of a sequenced portion of the tet(O)-mef(A) element from S. pyogenes strain m46 (EMBL accession no. AM492531) compared with the ORF map of the ...

These findings contribute to conclusively clarifying why, among S. pyogenes isolates with the M phenotype, the tetracycline-resistant ones are, as a rule, SmaI typeable, while the tetracycline-susceptible ones are not. Previous studies have shown that, while the two related genetic elements (Tn1207.3 and Φ10394.4) typical of tetracycline-susceptible isolates are inserted into the same prophage, the tet(O)-mef(A) element typical of tetracycline-resistant isolates is inserted into a different prophage (6). The present results show that the R-M cassette encoding, in Tn1207.3-Φ10394.4, the methyltransferase responsible for DNA resistance to cleavage by SmaI is absent in the tet(O)-mef(A) element where, together with an adjacent ORF, it is replaced by three new ORFs flanked, both upstream and downstream, by homologous regions.

Acknowledgments

This work was supported in part by a grant from the Italian Ministry of Education, University, and Research.

Footnotes

[down-pointing small open triangle]Published ahead of print on 11 June 2007.

REFERENCES

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