JAC Advance Access published online on May 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm120
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New Tn916-related elements causing erm(B)-mediated erythromycin resistance in tetracycline-susceptible pneumococci
Institute of Microbiology and Biomedical Sciences, Polytechnic University of Marche Medical School, 60020 Ancona, Italy
* Corresponding author. Tel: +39-071-2206294; Fax: +39-071-2206293; E-mail: pe.varaldo{at}univpm.it
Received 5 February 2007; returned 2 March 2007; revised 28 March 2007; accepted 28 March 2007
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Abstract |
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Objectives: To analyse the as yet unexplored genetic elements encoding erm(B)-mediated erythromycin resistance in tetracycline-susceptible pneumococci.
Methods: Sixteen Streptococcus pneumoniae clinical isolates sharing erm(B)-mediated erythromycin resistance and susceptibility to tetracycline were used. Gene detection was performed by PCR using both established and specially designed primers. S. pneumoniae R6, Streptococcus pyogenes 12RF and Enterococcus faecalis JH2–2 were used as recipients in mating experiments.
Results: Of the 16 test strains, 14 bore an unexpressed tet(M) gene which in 13 strains had a genetic linkage with erm(B). Three isolates yielded a 3.2 kb and 10 an 11.9 kb erm(B)/tet(M) amplicon. The former three showed genetic organizations similar to that of the composite element Tn3872, where the erm(B)-carrying Tn917 transposon is inserted into a Tn916-like element. Of the latter 10 isolates, 9 showed genetic organizations substantially overlapping with that of Tn6002, a newly sequenced erm(B)-containing Tn916-related transposon. The tenth isolate carried a novel composite element (designated Tn6003) resulting from the insertion into a Tn6002-like transposon of a fragment [designated macrolide–aminoglycoside–streptothricin (MAS) element] containing a second erm(B) (lacking the stop codon) and a variant of the aadE–sat4–aphA-3 cluster. The two tet(M)-negative isolates had different Tn3872-related elements, one containing a complete and one a deleted MAS fragment. Conjugative transfer was obtained from donors carrying Tn6002-related elements, not from donors carrying Tn3872-related elements.
Conclusions: In tetracycline-susceptible pneumococci with erm(B)-mediated erythromycin resistance, the erm(B) gene is carried on a variety of Tn916-related genetic elements either lacking tet(M) or, more often, carrying an unexpressed tet(M) gene.
Key Words: Streptococcus pneumoniae , genetic elements , resistance genes , Tn916 family conjugative transposons , erm(B) , tet(M)
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Introduction |
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In Streptococcus pneumoniae, the most common mechanism of resistance to macrolide, lincosamide and streptogramin B (MLS) antibiotics is a post-transcriptional methylase-mediated modification of 23S rRNA encoded by the erm(B) gene.1 erm(B) resistance can be expressed by pneumococci either constitutively (cMLS phenotype) or inducibly (iMLS phenotype).1,2
Among clinical S. pneumoniae isolates with erm(B)-mediated erythromycin resistance, most are also resistant to tetracycline (in Italy, 
90%).3 This association appears to reflect the widespread presence in pneumococcal populations of genetic elements (such as Tn15454 or Tn38725) resulting from the insertion of erm(B)-containing DNA into conjugative transposons of the Tn916 family, which typically carry tet(M).6
A much less-defined situation applies to the minority of erm(B)-positive pneumococci that are susceptible to tetracycline. Tn917, a small erm(B)-carrying transposable element discovered in Enterococcus faecalis,7 was subsequently also detected in pneumococci. In the latter, however, Tn917 was soon found to be inserted into a Tn916-like element to form the composite transposon Tn3872, originally identified in 12 isolates (11 resistant and 1 susceptible to tetracycline).5
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Materials and methods |
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Bacterial strains
All the strains (n = 16) susceptible to tetracycline (MIC, 
2 mg/L) were selected from a collection of 148 clinical isolates of erythromycin-resistant S. pneumoniae, carrying erm(B) as the sole erythromycin resistance gene, recovered in different areas of Italy in 2000–2002. Erythromycin MICs > 128 mg/L were recorded for all test strains, whose constitutive (cMLS) or inducible (iMLS) macrolide resistance phenotype was determined on the basis of the triple-disc (erythromycin, clindamycin and rokitamycin) test.2 All 16 test isolates were different strains based on a variety of typing characteristics (data not shown).
Antibiotics and susceptibility tests
Erythromycin and tetracycline MICs were determined by a standard agar dilution method.
Amplification experiments and gene detection
All primer pairs used in PCR experiments are listed in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)]. DNA preparation and amplification and electrophoresis of PCR products were carried out by established procedures as described elsewhere.8 The structure of the Tn916-related elements containing erm(B) was examined by PCR assays and sequence analysis using both established primers and primers specially designed from the sequence of Tn916 (accession no. U09422). Additional primers (J12 and J11) were synthesized based on the results of the erm(B)/tet(M) linkage.
DNA sequence analysis was performed by standard methods as described elsewhere.8
Mating assays were performed as described elsewhere,8 using S. pneumoniae test strains with different genotypic characteristics as donors and S. pneumoniae R6, Streptococcus pyogenes 12RF9 and E. faecalis JH2-2 as recipients.
Nucleotide sequence accession number
The sequenced portions, both containing a new macrolide–aminoglycoside–streptothricin (MAS) element, of the novel Tn916-related transposon Tn6003 and the Tn3872-related element SpnRi3erm(B) have been submitted to the GenBank Nucleotide Sequence Database and assigned accession nos. AM410044 and AM490850, respectively.
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Results and discussion |
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Phenotypic characterization of isolates
Based on their patterns of susceptibility to MLS antibiotics and the triple-disc test, 14 isolates were found to belong to the inducible (iMLS) and 2 to the constitutive (cMLS) macrolide resistance phenotype (Table 1). Tetracycline MICs ranged between 0.25 and 2 mg/L.
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Search for the tet(M) gene
Of the 16 tetracycline-susceptible test strains, 14 (the 2 cMLS isolates and 12/14 iMLS isolates) yielded a positive PCR with the tet(M) primers. The lack of tet(M) in the two negative strains was confirmed by Southern hybridization using a specific probe.
Search for an erm(B)/tet(M) linkage
In the 14 tet(M)-positive strains, the possible occurrence of a genetic linkage between erm(B) and the unexpressed tet(M) was first investigated by PCR. As shown in Table 1, a PCR product of 11.9 kb was obtained in nine iMLS isolates and one cMLS isolate by pairing primers ERMB1 and TETM3, whereas a smaller amplicon (3.2 kb) was obtained in two iMLS isolates and one cMLS isolate by pairing primers TETM2 and ERMB2. No linkage amplicon was obtained from the remaining iMLS isolate (strain Lt3).
Amplification experiments to detect Tn917 and Tn916 genes
To gain insights into the erm(B)-carrying genetic elements of the 16 test strains, we investigated them by PCR for the presence of two genes (tnpA and tnpR) associated with Tn91710 and of two genes (int and xis) associated with conjugative transposons of the Tn916 family6 (Table 1). A positive PCR reaction for the two Tn917 genes was obtained from five test strains: the three that yielded a 3.2 kb linkage amplicon and the two giving negative PCR and Southern hybridization reactions for tet(M). With regard to the two Tn916 genes, 14 isolates were positive for xis, and 12 also for int.
Genetic elements carrying erm(B) in tet(M)-positive isolates
Extensive PCR experiments with suitable primer pairs, performed to detect linkages between resistance and recombinase genes, and sequence analysis of the amplicons spanning erm(B) and tet(M) revealed various genetic organizations in the 13 test strains displaying a linkage between the two genes. The fourteenth tet(M)-positive isolate (Lt3), albeit bearing a silent tet(M) gene, showed no apparent erm(B)/tet(M) linkage or PCR evidence of any of the other genes tested (Table 1).
In the three isolates yielding the 3.2 kb (3203 bp) erm(B)/tet(M) linkage amplicon, the Tn917 transposon carrying erm(B) was inserted into orf9 of a Tn916-like element, at base 14 525 of the published sequence of Tn916. PCR assays showed that in these strains, Tn917 had the same orientation and was part of genetic structures (Table 1) resembling the composite transposon Tn3872.5
In the 10 isolates yielding the 11.9 kb (11 944 bp) erm(B)/tet(M) linkage amplicon, two different PCR products were obtained by analysing the regions flanking erm(B) using primers J12 and J11: one of 3.6 kb in the nine iMLS isolates, and one of 7.9 kb in the cMLS isolate (Ar4).
Sequence analysis of the 3.6 kb amplicon showed that erm(B) was located in a DNA fragment that included its leader peptide upstream of the gene and another, putative leader peptide and a transposase downstream of it. This fragment (2848 bp) lay between orf20 and orf19 of the Tn916 transposon at base 3847 of its published sequence. In these nine iMLS isolates, erm(B) and its flanking regions displayed 100% homology to the corresponding regions of the Tn916-related, erm(B)-containing transposon Tn6002 (accession no. AY898750). Further PCR and sequencing experiments showed that the entire genetic organization of the erm(B)-carrying element of these nine iMLS isolates overlapped with that of Tn6002, only strain Pt2 being int- and orf24-negative (Table 1).
Sequence analysis of the 7.9 kb amplicon obtained by pairing primers J12 and J11 in the cMLS isolate (Ar4) (accession no. AM410044) revealed that the genetic organization of the right portion of the amplicon substantially overlapped (99.5% homology) with that of the 3.6 kb amplicon described above. The remaining portion (4225 bp) appeared to be a rearrangement (99.4% homology) of part of plasmid pRE2511 of E. faecalis and contained, from upstream to downstream, another erm(B) gene (lacking the stop codon) with its leader peptide (98.9% homology), an aminoglycoside–streptothricin resistance cluster (aadE–sat4–aphA-3) (100% homology, save for a deletion of 511 nucleotides in aadE) and an ORF identical to orf47 of that plasmid (Figure 1a). This 4.2 kb fragment, that we designated MAS (macrolide–aminoglycoside–streptothricin) element, was inserted into orf20F of Tn6002, at base 4110 of its published sequence. A 222 bp sequence on the right end of the MAS element was homologous to the 3889–4110 Tn6002 region, corresponding to the end of orf20F and the intergenic region upstream of the erm(B) leader peptide. This particular sequence, absent in the Tn916 transposon, may have played a role in the insertion of the MAS element into Tn916-like transposons immediately upstream of the erm(B) leader peptide, as in the two tet(M)-negative isolates (see below). The new composite transposon (25 105 bp) found in S. pneumoniae Ar4—carrying tet(M), erm(B) and the MAS element—was designated Tn6003 (http://www.ucl.ac.uk/eastman/tn/) (Figure 1a).
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Sequence analysis of the DNA segments containing tet(M) and its regulatory region in the 13 test strains that displayed an erm(B)/tet(M) linkage showed 95% to 99% and 93% to 97% homology to the corresponding segments of Tn6002 and Tn916, respectively.
Genetic elements carrying erm(B) in tet(M)-negative isolates
In the two tet(M)-negative isolates (both iMLS), erm(B) was carried by transposon Tn917, which proved to have the same orientation and the same insertion into orf9 of a Tn916-like element as the three isolates yielding the 3.2 kb erm(B)/tet(M) linkage amplicon. Amplification and sequencing of the right and left junctions of the Tn917 transposon showed that, while the ORFs downstream of Tn917 matched those of Tn916, a completely different organization was detected upstream. In one strain (Ri3), a second erm(B) gene was detected in a fragment identical to the MAS element located immediately upstream of Tn917. The new element (16 692 bp) was identified as SpnRi3erm(B) (Figure 1b). In the other strain (Ap3), only the right portion of the MAS element (as far as aphA-3) was found upstream of Tn917. The new element (11 032 bp) was identified as SpnAp3erm(B) (Figure 1c).
Transferability of the composite elements
Five test strains were used as donors in mating experiments: Ro1 (carrying Tn6002); Ar4 (carrying Tn6003); Vt2 (carrying Tn3872); Ri3 [carrying SpnRi3erm(B)]; and Ap3 [carrying SpnAp3erm(B)]. No detectable transfer from any donor was obtained using S. pneumoniae R6 as the recipient. Also no detectable transfer to the other recipients was obtained from the three donors carrying Tn3872 or Tn3872-related elements, consistent with the originally reported non-transferability of Tn3872.5 In contrast, transfer was successful, albeit depending on the recipient used, from the two donors carrying Tn6002 or Tn6003: the former element was transferred from strain Ro1 only to S. pyogenes 12RF (frequency, 1.1 x 10–8), whereas the latter was transferred from strain Ar4 only to E. faecalis JH2-2 (frequency, 1.7 x 10–7).
This study shows that, in tetracycline-susceptible pneumococci with erm(B)-mediated macrolide resistance, the erm(B) gene can be carried by heterogeneous genetic elements. Interestingly, all these elements were related to Tn916, the prototype of a widespread family of conjugative transposons whose presence is typically associated with tetracycline resistance in Gram-positive bacteria.6 In these Tn916-related elements, the erm(B) gene was either detected in a Tn917 transposon inserted in Tn916 in a fashion reminiscent of the composite transposon Tn38725 or contained in Tn6002-like transposons. Except for the two isolates bearing Tn3872-related elements SpnRi3erm(B) and SpnAp3erm(B), all the other tetracycline-susceptible test strains (14/16) had unexpressed tet(M) genes. The paradoxical association between a tetracycline-susceptible phenotype and a tetracycline-resistant genotype suggests that transposons of the Tn916 family should no longer be regarded as being strictly associated with tetracycline resistance and may thus be more widespread in S. pneumoniae than currently believed.
Another interesting result of this study was the finding in pneumococci of a variant of the aminoglycoside–streptothricin resistance cluster so far described in staphylococci,12,13 enterococci11,14 and, more recently, in Campylobacter jejuni.15 In our strain Ar4, a new Tn6002-related composite element (Tn6003) contained a 4.2 kb fragment (MAS) formed by the aadE–sat4–aphA-3 gene cluster with a defective aadE determinant; upstream of this lay another erm(B) gene (lacking the stop codon), whereas an ORF identical to that (orf47) detected in the enterococcal plasmid pRE2511 was identified downstream of aphA-3. An identical MAS fragment was found immediately upstream of Tn917 in the Tn3872-related element [SpnRi3erm(B)] detected in one of our two tet(M)-negative S. pneumoniae strains; only the right portion of this element, with a deleted MAS fragment, formed the element [SpnAp3erm(B)] detected in the second tet(M)-negative strain.
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None to declare.
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Supplementary data |
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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Footnotes |
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Present address. Department of Biomedical Sciences and Technologies, Section of Medical Microbiology, University of Cagliari, Italy. 
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Acknowledgements |
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We are grateful to Eleonora Giovanetti, Andrea Brenciani and Alessandro Bacciaglia for helpful discussions. We also thank Adam P. Roberts for his qualified suggestions on naming the new genetic elements. This work was partly supported by the Italian Ministry of Education, University and Research.
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References |
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