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Antimicrobial Agents and Chemotherapy, April 2007, p. 1209-1216, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01484-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Elements Carrying erm(B) in Streptococcus pyogenes and Association with tet(M) Tetracycline Resistance Gene{triangledown}

Andrea Brenciani,1 Alessandro Bacciaglia,1 Manuela Vecchi,1 Luca A. Vitali,2 Pietro E. Varaldo,1* and Eleonora Giovanetti1

Institute of Microbiology and Biomedical Sciences, Polytechnic University of Marche, 60131 Ancona,1 Department of Molecular, Cellular and Animal Biology, Chair of Microbiology, University of Camerino, 62032 Camerino, Italy2

Received 24 November 2006/ Returned for modification 7 January 2007/ Accepted 16 January 2007


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ABSTRACT
 
This study was directed at characterizing the genetic elements carrying the methylase gene erm(B), encoding ribosome modification-mediated resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics, in Streptococcus pyogenes. In this species, erm(B) is responsible for MLS resistance in constitutively resistant isolates (cMLS phenotype) and in a subset (iMLS-A) of inducibly resistant isolates. A total of 125 erm(B)-positive strains were investigated, 81 iMLS-A (uniformly tetracycline susceptible) and 44 cMLS (29 tetracycline resistant and 15 tetracycline susceptible). Whereas all tetracycline-resistant isolates carried the tet(M) gene, tet(M) sequences were also detected in most tetracycline-susceptible isolates (81/81 iMLS-A and 7/15 cMLS). In 2 of the 8 tet(M)-negative cMLS isolates, erm(B) was carried by a plasmid-located Tn917-like transposon. erm(B)- and tet(M)-positive isolates were tested by PCR for the presence of genes int (integrase), xis (excisase), and tndX (resolvase), associated with conjugative transposons of the Tn916 family. In mating experiments using representatives of different combinations of phenotypic and genotypic characteristics as donors, erm(B) and tet(M) were consistently cotransferred, suggesting their linkage in individual genetic elements. The linkage was confirmed by pulsed-field gel electrophoresis and hybridization studies, and different elements, variably associated with the different phenotypes/genotypes, were detected and characterized by amplification and sequencing experiments. A previously unreported genetic organization, observed in all iMLS-A and some cMLS isolates, featured an erm(B)-containing DNA insertion into the tet(M) gene of a defective Tn5397, a Tn916-related transposon. This new element was designated Tn1116. Genetic elements not previously described in S. pyogenes also included Tn6002, an unpublished transposon whose complete sequence is available in GenBank, and Tn3872, a composite element resulting from the insertion of the Tn917 transposon into Tn916 [associated with a tet(M) gene expressed in some cMLS isolates and silent in others]. The high frequency of association between a tetracycline-susceptible phenotype and tet(M) genes suggests that transposons of the Tn916 family, so far typically associated solely with a tetracycline-resistant phenotype, may be more widespread in S. pyogenes than currently believed.


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INTRODUCTION
 
A considerable and rapid increase in the rate of erythromycin resistance among Streptococcus pyogenes isolates has been reported in Italy in the past decade. An overall incidence around 43%, i.e., the highest recorded since a Japanese epidemic in the 1970s (21), was reported in an extensive 2-year nationwide survey carried out in the late 1990s (35). Detailed epidemiological investigations confirmed the phenotypic and genotypic heterogeneity of S. pyogenes isolates resistant to macrolide, lincosamide, and streptogramin B (MLS) antibiotics (10, 16). In these strains, resistance may be due to efflux or target site modification mechanisms. In the former case, it is mediated by the mef(A) gene (6) and is associated with a special resistance pattern (M phenotype) characterized by resistance, among MLS antibiotics, only to 14- and 15-membered macrolides and usually at a low level (33). In the latter case, besides occasional mutations in 23S rRNA or ribosomal proteins, posttranscriptional target site modifications are caused by erm class methylases that modify an adenine residue in the 23S rRNA (36) and are associated with a constitutive (cMLS) or inducible (iMLS) resistance phenotype. Whereas in cMLS S. pyogenes isolates the methylase gene is consistently erm(B), iMLS isolates have been further subdivided into three distinct types. One (iMLS-A) also associated with the erm(B) gene, and two (iMLS-B and iMLS-C) associated with the more recently described erm(A), originally designated erm(TR) (29). iMLS-A, iMLS-B, and iMLS-C are distinguished by the features and level of their resistance to 14-, 15-, and 16-membered macrolides and to ketolides (15, 16) and are easily differentiated in the laboratory by a triple-disk test (16).

Several new findings have also emerged from recent studies of the association between erythromycin and tetracycline resistance in S. pyogenes. In the tetracycline-resistant isolates of either the M phenotype or the iMLS-B/iMLS-C phenotypes, the tetracycline resistance determinant is invariably tet(O), which appears to be linked, respectively, to the erythromycin resistance determinants mef(A) or erm(A) (13). Extensive studies of the former linkage led to the discovery of a unique class of tet(O)-mef(A) elements resulting from the insertion of both the tet(O) gene and a variable mef(A)-carrying Tn1207.1-related transposon into an element of prophage origin (4, 14). Among the S. pyogenes isolates with erm(B)-mediated erythromycin resistance, different situations are found in those of the iMLS-A phenotype, which are consistently tetracycline susceptible (16, 27), and in those of the cMLS phenotype. Of the latter, the tetracycline-resistant isolates bear tet(M) as the tetracycline resistance determinant (13), typically linked to erm(B) in streptococci, in a variety of elements (such as Tn1545, Tn3703, Tn3704, Tn3872) resulting from the insertion of erm(B)-containing DNA into conjugative transposons of the Tn916 family (7, 20, 26).

It has long been held that, in streptococci, erm(B) usually has a chromosomal location, but it may also be located on plasmids (17). However, most available data have been established long ago from Streptococcus species mostly other than S. pyogenes, and in more recent studies, the focus has largely been on Streptococcus pneumoniae (18). This study was directed at characterizing the genetic elements carrying erm(B) and their linkage with tetracycline resistance determinants in a large collection of S. pyogenes isolates. A notable finding of this study was that, in S. pyogenes isolates with erm(B)-mediated erythromycin resistance, tetracycline susceptibility is often associated with the presence of silent tet(M) genes.


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MATERIALS AND METHODS
 
Bacteria. A total of 125 clinical isolates of erythromycin-resistant S. pyogenes, all carrying erm(B) as the sole erythromycin resistance gene, were investigated. Strain identification was performed using bacitracin disks (Oxoid Ltd., Basingstoke, England) and a latex agglutination assay (Strep Plus; Oxoid). Erythromycin MICs of >128 µg/ml were recorded for all test strains, whose cMLS or iMLS-A macrolide resistance phenotype was determined on the basis of patterns of susceptibility to MLS antibiotics and the triple-disk (erythromycin, clindamycin, and josamycin) test (16). All isolates (the vast majority of which were from throat swab cultures of children) were collected between 1997 and 2004 from several Italian laboratories and represented geographic and chronologic diversities, i.e., isolates exhibiting either macrolide resistance phenotype (cMLS or iMLS-A) had been isolated in different towns and/or years.

Antibiotics and susceptibility tests. Erythromycin and tetracycline were purchased from Sigma Chemical Co., St. Louis, MO. Broth microdilution MICs were determined as recommended by the Clinical and Laboratory Standards Institute (8). S. pneumoniae ATCC 49619 was used for quality control. In some isolates, tetracycline MICs were also determined after induction with low levels of tetracycline (12). Briefly, for each strain, a few colonies from an overnight growth on brain heart infusion agar (Oxoid) were used to inoculate two brain heart infusion agar plates, one unsupplemented (no induction control) and one supplemented with subinhibitory tetracycline (0.05 µg/ml). Cells were harvested after no longer than 20 h of growth and were used for MIC evaluation.

Gene detection and amplification experiments. All primer pairs used in PCR experiments are listed in Table 1. DNA preparation and amplification and electrophoresis of PCR products were carried out by established procedures and following recommended conditions for the use of individual primer pairs. The Ex Taq system (TaKaRa Bio, Shiga, Japan) was used in the amplification experiments expected to yield PCR products exceeding 3 kb in size. To determine and characterize a linkage between erm(B) and tet(M), PCR assays were performed using one primer specific for a region within the former gene and another specific for a region within the latter gene; the four primer combinations associated with the possible reciprocal orientation of the two genes were analyzed. When amplicons spanning erm(B) and tet(M) were obtained, PCR products were purified using Montage PCR filter units (Millipore Corporation, Bedford, MA) and sequenced.


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  		TABLE 1. Oligonucleotide primer pairs used

Plasmid analysis. Plasmid isolation was performed as described by O'Sullivan and Klaenhammer (24).

Mating experiments. S. pyogenes test strains with different combinations of phenotypic and genotypic characteristics were used as donors. S. pyogenes 12RF, a clinical strain selected in the laboratory for rifampin (25 µg/ml) and fusidic acid (25 µg/ml) resistance and susceptible to both erythromycin (MIC, ≤0.015 µg/ml) and tetracycline (MIC, ≤0.015 µg/ml), was used as the recipient. Conjugal transfer was performed on a membrane filter (37). The frequency of transfer was expressed as the number of transconjugants per recipient. Selected transconjugants were verified as carrying the erm(B) and tet(M) genes. Mating experiments were done at least three times.

PFGE analysis. Macrorestriction with SmaI endonuclease (New England Biolabs, Beverly, MA) and pulsed-field gel electrophoresis (PFGE) analysis were performed as described elsewhere (27).

Southern blotting and hybridization. DNA fragments generated by PFGE analysis were transferred to nylon membranes (Bio-Rad Laboratories, Richmond, CA) by capillary transfer and hybridized with [{alpha}-32P]dCTP-labeled probes specific for the erm(B) and tet(M) genes. These probes were obtained by PCR with the oligonucleotide primers reported in Table 1.

Restriction enzymes. Besides SmaI, used for PFGE analysis, restriction endonucleases EcoRV, HindIII, MfeI, and NdeI (New England Biolabs) were used for specific amplicon digestion assays.

DNA sequence analysis. Amplicon sequencing was carried out bidirectionally using ABI Prism (Perkin-Elmer Applied Biosystems, Foster City, CA) with dye-labeled terminators. Sequences were analyzed using the Sequence Navigator software package (Perkin-Elmer Applied Biosystems). Open reading frame (ORF) analysis was performed with the DNA Star software package (Lasergene, Madison, Wis.), and sequence similarity and conserved domain searches were carried out using the tools (BLAST and CDART) available online at the National Center for Biotechnology Information of the National Library of Medicine (Bethesda, MD) (http://www.ncbi.nlm.nih.gov).

Induction assays with mitomycin C. Experiments to recognize a possible prophage association of specific genetic elements carrying erm(B) and tet(M) were performed as described elsewhere (14), with induction with mitomycin C monitored by PCR using primer pairs targeting the two resistance genes.

Nucleotide sequence accession numbers. The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL sequence database and assigned accession numbers AM411377, AM411624, and AM411452.


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RESULTS AND DISCUSSION
 
Phenotypic and genotypic characterization of isolates. Of the 125 erm(B)-positive clinical strains tested, independently isolated in Italy over the past decade and representing geographic and chronologic diversities, 81 were assigned to the iMLS-A phenotype and 44 to the cMLS phenotype. In line with previous studies of the prevalence of tetracycline resistance in erythromycin-resistant S. pyogenes isolates in Italy (16, 27), the 81 iMLS-A isolates were found to be uniformly susceptible to tetracycline (MIC range, ≤0.125 to 0.25 µg/ml), whereas of the 44 cMLS isolates, 29 (66%) were tetracycline resistant (MIC range, 32 to 128 µg/ml) and 15 (34%) were tetracycline susceptible (MIC range, ≤0.125 to 0.25 µg/ml).

The 29 tetracycline-resistant cMLS isolates, investigated by PCR for the presence of tetracycline resistance determinants tet(M), tet(O), tet(K), and tet(L), were all found to carry tet(M). Unexpectedly, one tetracycline-susceptible iMLS-A isolate run as a negative control yielded a positive reaction for the tet(M) gene. This led us to extend the PCR test for tet(M) to all tetracycline-susceptible test strains. Surprisingly, all of the 81 iMLS-A isolates and 7/15 tetracycline-susceptible cMLS isolates yielded a positive reaction. Neither the 7 tetracycline-susceptible tet(M)-positive cMLS isolates nor 14 isolates randomly selected from the 81 iMLS-A isolates showed any variation in their tetracycline MICs after induction. These findings suggested that the tet(M) was present in a silent form in all of these tetracycline-susceptible but tet(M)-positive isolates.

Initial characterization of erm(B)-carrying genetic elements for the presence of Tn916-related transposons or Tn917. Fifty erm(B)- and tet(M)-positive isolates (including the 14 randomly selected isolates of the iMLS-A phenotype and the 29 tetracycline-resistant and 7 tetracycline-susceptible isolates of the cMLS phenotype) were tested by PCR for the presence of three genes associated with conjugative transposons of the Tn916 family: the conventional int (integrase) and xis (excisase) genes (7) and the tndX (resolvase) gene, which replaces int and xis in the Tn916-related transposon Tn5397, originally found in Clostridium difficile (22, 28). As shown in Table 2, five phenotype/genotype (P/G) combinations (designated A to E) were detected. All of the 14 iMLS-A isolates harbored tndX (P/G combination A). Of the 29 tetracycline-resistant cMLS isolates, 21 had both int and xis (P/G combination B) and 8 had only xis (P/G combination C); of the 7 tetracycline-susceptible cMLS isolates, 4 had xis (P/G combination D) and 3 had tndX (P/G combination E).


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  		TABLE 2. Characterization of 58 S. pyogenes test strains carrying erm(B) as the sole erythromycin resistance gene, subdivided according the P/G combinations displayed

The 8 tetracycline-susceptible isolates of the cMLS phenotype that yielded a negative PCR with tet(M) primers were confirmed not to carry tet(M) by Southern blotting and were investigated for the presence of Tn917, a well-known transposable element carrying erm(B) (30). Using specific primers targeting the tnpA (transposase) and tnpR (resolvase) genes of Tn917 (25), negative PCRs were obtained for 6 (P/G combination F) and positive reactions were obtained for 2 (P/G combination G) of these 8 isolates (Table 2). Positive PCRs with these Tn917-specific primers were also obtained for the strains displaying P/G combinations C and D (Table 2).

Conjugal transfer of the erm(B) and tet(M) genes, PFGE analysis, and hybridization experiments. Seven test strains, chosen as representative of the different P/G combinations, were used as donors in mating experiments (Table 3). erm(B) and tet(M) were cotransferred to the recipient from all of the 5 tet(M)-positive donors (from donor strain B-83, only when erythromycin was used for selection), suggesting a linkage of the two resistance genes in individual genetic elements. The highest transfer frequencies were recorded from the 2 tndX-positive donors (P/G combinations A and E). It is worth noting that, when transferred from tetracycline-susceptible donors, tet(M) was still unexpressed in the transconjugants which, like the donors, were tetracycline susceptible and exhibited no MIC variation after induction. No transconjugants were obtained from the 2 tet(M)-negative cMLS donors (P/G combinations F and G).


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  		TABLE 3. Conjugal transfer of resistance genes erm(B) and tet(M) from S. pyogenes donors displaying different P/G combinations to the same susceptible S. pyogenes recipient

The linkage between erm(B) and tet(M) was confirmed by PFGE and hybridization studies (Table 3). Transconjugants receiving both erm(B) and tet(M) had PFGE patterns showing insertion of new DNA (usually ranging from ca. 21 to over 50 kb), consistent with the linkage and chromosomal location of the two genes. Likewise, hybridization experiments indicated that the extra bands (i.e., the new larger fragments) hybridized with both a tet(M)-specific and an erm(B)-specific probe. The variability of new DNA insertions suggested the involvement of a number of different genetic elements carrying erm(B) and tet(M).

Plasmid analysis and hybridization assays in tet(M)-negative isolates. In the 2 tet(M)-negative cMLS isolates with PCR evidence of Tn917 ORFs (P/G combination G), the erm(B)-specific probe hybridized with a band below the chromosome, consistent with a plasmid location of this transposon (17). However, the plasmid was not easily detectable on the plasmid screens. Plasmid DNA could not be isolated from any of the 6 tet(M)-negative cMLS isolates with no PCR evidence of Tn917 ORFs (P/G combination F), and in these cases, the erm(B)-specific probe hybridized with the chromosomal band.

DNA amplification and sequencing in tet(M)-positive isolates. PCR and sequencing experiments were carried out to substantiate and characterize the erm(B)/tet(M) linkage in representative isolates of the different P/G combinations (A to E), yielding a positive PCR with tet(M) primers (Table 2).

(i) P/G combination A. With all 14 strains tested, a PCR product (ca. 2.7 kb) was obtained only by pairing primers ERMB2 and TETM3. DNA sequencing confirmed the presence of erm(B) on the left end and that of tet(M), oriented opposite to erm(B), on the right end of the amplified sequence (Fig. 1). tet(M) was part of a Tn5397-related element, as demonstrated by the expected distance (2,462 bp) between tet(M) and tndX. Whereas the ORFs upstream of tet(M) in Tn5397 were not detected by DNA amplification, DNA sequencing confirmed that the region downstream of tet(M) matched the one of the Tn5397 element. However, this tet(M) gene was incomplete, owing to the insertion of an erm(B)-containing DNA fragment in its coding sequence, at base 15,019 of the published sequence of Tn5397 (accession no. AF333235). Between tet(M)* and erm(B), a 687-bp ORF, having the same direction of transcription as erm(B) and identical to the enterococcal transposase IS1216 (19), was encompassed by two inverted repeat sequences. The new composite structure (ca. 50 kb) apparently resulted from the insertion of erm(B)-containing DNA, of likely enterococcal origin, into the tet(M) gene of a defective Tn5397 and was designated Tn1116. Induction assays with mitomycin C excluded that this new element was prophage associated.


Figure 1
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  		FIG. 1. Schematic representation of Tn1116, a novel Tn916 family transposon resulting from the insertion of erm(B)-containing DNA into the tet(M) gene of a defective Tn5397. The unsequenced region is tentatively represented upstream of the sequenced region. A squared arrow indicates erm(B); a striped arrow indicates tet(M). The distance between erm(B) and tet(M) was 1,090 bp. Thin arrows below erm(B) and tet(M) indicate the positions and directions of the primers used to investigate the linkage between the two resistance genes. The inverted repeat sequence (IR) upstream of the IS1216 transposase was incomplete. The nucleotide sequence of the DNA region between the erm(B) gene and the tndX gene has been deposited under accession no. AM411377.

(ii) P/G combination B. With all 21 test strains, a PCR product (ca. 12 kb) was obtained only by pairing primers ERMB1 and TETM3. DNA sequencing confirmed the presence of erm(B) and tet(M) on the left and right end, respectively, of the amplified sequence (Fig. 2). The distance between erm(B) and tet(M) was ca. 9.9 kb. Digestion of the amplicon with restriction enzymes yielded DNA fragments fully consistent with Tn6002, a Tn916-related, erm(B)-containing unpublished transposon detected in Streptococcus cristatus (previously designated Tn916Erm) whose complete sequence is available in GenBank (accession no. AY898750). The endonucleases used (and the sizes of the fragments obtained) were as follows: EcoRV, 10,713, 892, and 321 bp; HindIII, 10,672 and 1,254 bp; MfeI, 10,496, 1,015, 386, and 29 bp; and NdeI, 1,084 and 10,842 bp. Using primer pairs designed in our laboratory (Table 1) that enable amplification of DNA segments spanning the whole Tn916 (M. P. Montanari, I. Cochetti, E. Tili, and P. E. Varaldo, unpublished data), a full correspondence with the Tn6002 organization was apparent.


Figure 2
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  		FIG. 2. Schematic representation of the linkage between genes erm(B) (squared arrow) and tet(M) (striped arrow) in isolates displaying P/G combination B. The genetic organization was fully consistent with that of Tn6002, a Tn916-related, erm(B)-containing unpublished transposon (formerly called Tn916Erm) whose complete sequence is available in GenBank (accession no. AY898750). ORFs other than tet(M) that are shared by Tn6002 and Tn916 are represented as gray arrows; ORFs other than erm(B) that are not present in Tn916 are represented as black arrows. Black bars above the corresponding ORFs indicate the amplicons (whose sizes are expressed in bp) obtained with the primers whose sequences are reported in Table 1. Thin arrows below erm(B) and tet(M) indicate the positions and directions of the primers used to investigate the linkage between the two resistance genes.

(iii) P/G combination C. With all 8 test strains, a PCR product (ca. 3 kb) was obtained only by pairing primers TETM2 and ERMB2. DNA sequencing, carried out for the entire PCR product, confirmed the presence of tet(M) and erm(B) on the left and right end, respectively, of the amplified sequence (Fig. 3). The distance between tet(M) and erm(B) was 1,102 bp. The genetic organization revealed by DNA sequencing was found to overlap with that of Tn3872, a composite element resulting from the insertion of the Tn917 element into Tn916 (20, 25). Exactly as in Tn3872, the left region of Tn917 was inserted into orf9 of a Tn916-like element, at base 14,525 of the published sequence of Tn916 (accession no. U09422). Using the above-mentioned primer pairs designed in our laboratory (enabling amplification of DNA segments spanning the whole Tn916), all ORFs of Tn3872 except orf24 and int, i.e., the two located on the left and right end of the transposon, respectively, were detected. While the absence of orf24 has been documented in other Tn916-related elements like Tn5397 (28), the lack of PCR evidence of the integrase gene could depend on an int variant not detectable by the int primers.


Figure 3
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  		FIG. 3. Schematic representation of the linkage between genes erm(B) (squared arrow) and tet(M) (striped arrow) in isolates displaying P/G combinations C and D. The genetic organization was consistent with that of Tn3872, a composite element resulting from the insertion of the Tn917 element (ca. 5.3 kb) into Tn916 (ca. 18 kb), except for the lack of PCR evidence of the two genes (orf24 and int) located on the left and right end of the transposon. ORFs of Tn916 other than tet(M) are represented as gray arrows (white arrows with dashed outlines indicate ORFs undetected by PCR). ORFs of Tn917 other than erm(B) are represented as black arrows. Black bars above the corresponding ORFs indicate the amplicons (whose sizes are expressed in bp) obtained with the primers whose sequences are reported in Table 1. Thin arrows below erm(B) and tet(M) indicate the positions and directions of the primers used to investigate the linkage between the two resistance genes. The nucleotide sequence of the DNA region between the tet(M) gene and the erm(B) gene has been deposited under accession no. AM411624.

(iv) P/G combination D. The genetic organization detected by amplification and sequencing assays in the 4 strains displaying this P/G combination closely resembled that described above (Fig. 3) for the 8 isolates displaying P/G combination C, i.e., the same genotype [erm(B) tet(M) xis] and the same macrolide resistance phenotype (cMLS) but resistance instead of susceptibility to tetracycline. With these 4 strains, a PCR product (ca. 1.4 kb) was obtained by pairing primers M1 and O7, used to detect the regulatory region upstream of tet(M). DNA sequencing (accession no. AM411452) revealed the presence of point mutations in the sequence encoding the leader peptide, which might be related to the silencing of the tet(M) gene.

(v) P/G combination E. The genetic organization detected by amplification and sequencing assays in the 3 strains displaying this P/G combination was comparable with the one described above for Tn1116 (Fig. 1) in the 14 strains (P/G combination A) exhibiting the inducible rather than the constitutive macrolide resistance phenotype but showing the same genotype [erm(B) tet(M) tndX] and sharing a distinctive association between a tetracycline-susceptible phenotype and a tetracycline-resistant genotype. Induction assays with mitomycin C again excluded a prophage association.

Knowledge of the mechanisms and genetics of macrolide resistance in streptococci has greatly advanced over the last decade. A much greater amount of experimental work, however, has been focused on the emerging topic of efflux-mediated resistance rather than on the more conventional 23S rRNA methylation-mediated resistance. Furthermore, among erm class methylase genes of S. pyogenes, greater attention has been paid to erm(A) than to the classic, long-established erm(B) gene. The present study, while readdressing the classic subject of erm(B)-mediated erythromycin resistance in S. pyogenes, enabled us to highlight the marked heterogeneity of the genetic elements carrying erm(B) in this species, and an association with the tetracycline resistance determinant tet(M) that was more widespread and complex than expected.

Of the variable genetic elements carrying erm(B) in S. pyogenes, aside from a few plasmid locations, most were either previously unknown, like Tn1116, the new defective Tn5397 derivative with the erm(B)-IS1216 inserted into tet(M), or not previously described for S. pyogenes. The latter included Tn6002, an unpublished transposon whose complete sequence is available in GenBank, and Tn3872, a composite element resulting from the insertion of the Tn917 transposon into Tn916 (20, 25).

Such heterogeneity was even more noticeable in light of the unique but highly variable association with the tet(M) gene, which in either an expressed or a silent form was detected in the vast majority of erm(B)-carrying isolates. A linkage between erm(B) and tet(M) is well known in streptococci, mainly due to the widespread occurrence in these organisms of genetic elements resulting from the insertion of erm(B)-containing DNA into conjugative transposons of the Tn916 family, which typically carry tet(M) (7, 26). Tn1545, Tn3703, Tn3704, and Tn3872 (7, 20, 26) are well-known examples of such erm(B)/tet(M) elements. However, our findings disclose a more complex situation for S. pyogenes. The above-mentioned tet(M) gene homologous to that of Tn5397, inactivated by the insertion of erm(B)-containing DNA into its coding sequence, was identified in all iMLS-A isolates as well as in a few cMLS isolates. In other cMLS isolates sharing a genetic organization closely resembling that of Tn3872, the tet(M) gene was either expressed or silent, probably depending on the presence of a normal or altered regulatory region. However, all these variable genetic elements were related to Tn916. It is worth noting that a tetracycline-susceptible tet(M)-positive isolate was reported in the study where Tn3872 was first described for S. pneumoniae (20) and that different tet(M) variants, some associated with tetracycline resistance and some with tetracycline susceptibility, have been detected in Tn916-like elements recently found in clinical isolates of Clostridium difficile (31). Remarkably, in some S. pneumoniae isolates, Tn916-like elements have also been found to carry erythromycin efflux resistance genes like mef(E) (11) and mef(I) (9; M. Mingoia, P. E. Varaldo, and M. P. Montanari, unpublished data). Unlike genetic elements carrying the mef(A) efflux gene in S. pyogenes, which have been shown to be all chimeric structures (3, 14), no prophage association was documented in the same species for elements carrying erm(B). The unsuspected high frequency of an association between a tetracycline-susceptible phenotype and the presence of a tet(M) genotype suggests that transposons of the Tn916 family, so far typically associated with a tetracycline-resistant phenotype, may in fact be more widespread in S. pyogenes than currently believed.


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ACKNOWLEDGMENTS
 
We are grateful to Maria Pia Montanari, Ileana Cochetti, Emily Tili, and Marina Mingoia for helpful discussions and for permission to use a set of Tn916-specific primers as well as to mention some of their unpublished data. We also thank Marilyn C. Roberts for her invaluable collaboration in the early experiments suggesting an association between a tetracycline-susceptible phenotype and a tetracycline-resistant genotype in S. pyogenes, Adam P. Roberts for his qualified suggestions on transposon naming, and Lucia Padella for her excellent assistance.

This work was partly supported by the Italian Ministry of Education, University and Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Microbiology and Biomedical Sciences, Polytechnic University of Marche Medical School, Via Tronto 10/A, 60020 Ancona, Italy. Phone: 39 071 2206294. Fax: 39 071 2206293. E-mail: pe.varaldo{at}univpm.it Back

{triangledown} Published ahead of print on 29 January 2007. Back


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Antimicrobial Agents and Chemotherapy, April 2007, p. 1209-1216, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01484-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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