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Antimicrobial Agents and Chemotherapy, October 2006, p. 3361-3366, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00277-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The mef(E)-Carrying Genetic Element (mega) of Streptococcus pneumoniae: Insertion Sites and Association with Other Genetic Elements

Maria Del Grosso,1 Romina Camilli,1 Francesco Iannelli,2 Gianni Pozzi,2 and Annalisa Pantosti1*

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome,1 LAMMB, Dipartimento di Biologia Molecolare, Università di Siena, Siena, Italy2

Received 4 March 2006/ Returned for modification 5 June 2006/ Accepted 6 August 2006


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ABSTRACT
 
The structure of the macrolide efflux genetic assembly (mega) element, its genomic locations, and its association with other resistance determinants and genetic elements were investigated in 16 Streptococcus pneumoniae isolates carrying mef(E), of which 1 isolate also carried tet(M) and 4 isolates also carried tet(M) and erm(B). All isolates carried a mega element of similar size and structure that included the operon mef(E)-msr(D) encoding the efflux transport system. Among tetracycline-susceptible isolates, six different integration sites were identified, five of which were recognized inside open reading frames present in the R6 genome. In the five isolates also carrying tet(M), mega was inserted in different genetic contexts. In one isolate, it was part of previously described Tn916-like element Tn2009. In another isolate, mega was inserted in a transposon similar to Tn2009 that also included an erm(B) element. This new composite transposon was designated Tn2010. Neither Tn2009 nor Tn2010 could be transferred by conjugation to pneumococcal or enterococcal recipients. In the three isolates in which mega was not physically linked with tet(M), this gene was associated with erm(B) in transposon Tn3872, a Tn916-like element. Homologies between the chromosomal insertions of these composite transposons and sequences of multidrug-resistant pneumococcal genomes in the databases indicate the presence of preferential sites for the integration of composite Tn916-like elements carrying multiple resistance determinants in S. pneumoniae.


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INTRODUCTION
 
Macrolide resistance in Streptococcus pneumoniae is mediated by two main mechanisms: target modification due to a ribosomal methylase encoded by the erm(B) gene, which confers high-level resistance to macrolides, lincosamides, and streptogramin B (21), and an efflux transport system associated with the mef gene, which confers resistance to 14- and 15-membered macrolides only (6, 35). Molecular epidemiology studies have shown that the prevalence of these two mechanisms among macrolide-resistant pneumococci varies in different geographical areas. In the United States (18), Canada (15), and the United Kingdom (2), the efflux system is more common, while in Spain (34), Italy (24), and other European countries (29), the ribosomal modification is prevalent. In addition, isolates carrying both resistance determinants are becoming increasingly common in several areas of the globe (12, 19, 23) and have also been found, although rarely, in Italy (24, 25).

mef, originally considered the necessary and sufficient determinant of the efflux mechanism, appears to be a component of an efflux system that includes mef and the associated msr(A) homolog, recently renamed mrs(D) (8, 35). mef encodes two transmembrane domains, and msr(D) encodes two ATP-binding domains, likely constituting an efflux transport system of the ATP-binding cassette superfamily (F. Iannelli, M. Santagati, J. D. Doquier, M. Cassone, M. R. Oggioni, G. Rossolini, S. Stefani, and G. Pozzi, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1188, 2004) (1). Two subclasses of the mef gene have been found in pneumococci, mef(A) and mef(E), which share 90% nucleotide identity. Recently, a new variant designated mef(I) has been described in two pneumococcal isolates from Italy (7).

In Italy, mef(A) is the predominant efflux-associated gene, while mef(E) is found in approximately 15% of S. pneumoniae strains carrying the efflux pump (unpublished data). In other geographical areas, such as North America and Asia, mef(E) is by far the most common gene associated with macrolide efflux in pneumococci (8).

In S. pneumoniae, mef(A) and mef(E) are carried by two similar but distinct elements, designated, respectively, Tn1207.1 and the mega (macrolide efflux genetic assembly) element (13, 33). Tn1207.1 is a defective transposon of 7.2 kb that corresponds to the left (L) end of the larger Tn1207.3 transposon that carries mef(A) in Streptococcus pyogenes (32). In pneumococci, Tn1207.1 is always found inserted at the same specific chromosomal site, corresponding to the competence gene celB (2, 9, 33). The population structure of isolates carrying Tn1207.1 is clonal, and almost all of the isolates belong to serotype 14 and to the internationally disseminated clone England14-9 (9, 38). The mega element, 5.5 kb in size, is similar to Tn1207.1 but lacks the region upstream of mef. The mega element can be found inserted at different chromosomal sites (9, 13) in strains belonging to different serotypes and clonal groups (9, 31, 38). Recently, we reported that mega can be part of Tn2009, a composite element where mega is integrated in orf6 of a Tn916-like transposon carrying tet(M) (10).

In this study, we have examined the mega element in several S. pneumoniae isolates to characterize its structure, termini, and insertion sites. We have found new mega insertion sites and a new composite element, designated Tn2010, that is similar to Tn2009 but also contains the erm(B) gene.


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MATERIALS AND METHODS
 
Bacterial strains. Fifteen S. pneumoniae isolates carrying mef(E) were derived from a collection of about 1,000 strains obtained in the course of different surveillance programs carried out in Italy from 1997 to 2004 (24). These isolates represented all of the mef(E)-positive strains in the collection, with the exception of two strains previously described (10). One additional isolate, PGX1416, was originally obtained from Glaxo, Verona, Italy (24). PN150, carrying Tn2009 (10), was the control strain in the PCR mapping assays. S. pneumoniae FP10 and Enterococcus faecalis JH2-2 were used as recipients (16, 17) and DP1322, carrying Tn5251 (28), was used as a control in conjugation experiments.

Characterization of isolates. Antibiotic susceptibilities, serotyping, and detection of mef(E), tet(M), and erm(B) were performed as previously described (14, 24). Genetic relatedness among the isolates was studied by pulsed-field gel electrophoresis (PFGE) of SmaI-digested bacterial DNAs. PFGE types and subtypes were defined as previously described (14).

Mapping of mega and detection of mega insertion sites. Genomic DNA of the pneumococcal isolates was prepared with a commercial kit (MasterPure DNA purification kit; Epicenter, Madison, Wisconsin).

The genetic structure of mega was examined by PCR mapping with three sets of primer pairs to generate three overlapping fragments (Table 1 and Fig. 1). The intergenic region between mef(E) and msr(D) was examined with primers MEF5 (Table 1) and MEF4 (9). The sizes of the fragments obtained with the different isolates were compared with those obtained with PN150 (10).


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  		TABLE 1. Oligonucleotide primers used in this study to characterize mega and its chromosomal insertion sites


Figure 1
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  		FIG. 1. Genetic structure of mega. The element is boxed, and the chromosome is represented by a dotted line. The positions of the primers used for PCR mapping, for iPCRs, and to define the junctions of mega are indicated by arrowheads. The expected amplicons for PCR mapping are indicated by bars. The primers used to amplify the LJ and RJ of mega for each insertion site are collectively indicated as primers annealing upstream (U primer) and downstream (D primer) of the insertion site.

The L junction (LJ) and right (R) junction (RJ) of mega were determined by using two inverse PCR (iPCR) assays. To define the LJ, EcoRI-restricted genomic DNA was ligated and used as the template in an iPCR assay with primer pair SG5-MEF5 (Table 1 and Fig. 1). To define the RJ, PstI-restricted genomic DNA was used in a similar iPCR with primer pair OM1-SG1 (Table 1 and Fig. 1). After sequencing of the amplicons obtained, two primers specific for each insertion site were chosen, annealing, respectively, upstream (U primer) or downstream (D primer) of the insertion of mega (Table 1 and Fig. 1). In each isolate, the junctions of mega were confirmed by direct PCR assays with two pairs of primers: for the LJ, a U primer and OM18, annealing to mega; for the RJ, OM15, annealing to mega and a D primer (Table 1 and Fig. 1).

In isolates carrying mef(E) and tet(M), the location of mega in Tn2009 was investigated by PCR with primer pairs SG3-OM18 and SG1-TetMd (10, 14). Sequencing was performed to confirm the insertion sites at the nucleotide level.

Structure of the composite elements and detection of chromosomal insertion sites. PCR mapping of Tn916-like elements was performed as previously described (10). The sizes of the fragments obtained were compared with those obtained by using the genomic DNA of PN150.

The DNA regions flanking the ends of the composite elements were explored by two iPCR assays. The LJ was determined as previously described (10). The RJ was determined by a similar iPCR with NdeI-restricted genomic DNA and primers TN1 (5'-ATAAAGTGTGATAAGTCCAG-3') and TN2 (5'-GAAGTAGAAGCTAAAGATGG-3'), designed on the basis of the Tn916 sequence (GenBank accession no. U09422). The results were confirmed by direct amplification of the junctional fragments and sequencing.

In isolates carrying erm(B), the location of this gene inside the composite transposons was investigated by PCR with different combinations of primers EB1 and EB2, annealing to erm(B) (14), with primers annealing to Tn916 (27).

Conjugation experiments. To test the transferability of the composite elements, two different recipient strains were used in mating experiments: S. pneumoniae FP10 (16) and E. faecalis JH2-2 (17). Donor bacteria were grown in the presence of either tetracycline (5 µg/ml) or erythromycin (1 µg/ml), and mating experiments were carried out as previously described (9). Transconjugants were selected by using plates containing the antibiotics suitable to select the respective recipients (10, 17) and either erythromycin (1 µg/ml) or tetracycline (5 µg/ml). The conjugation assays were replicated three times.

Nucleotide sequence accession numbers. The sequences of the LJ and RJ of Tn2010 in PGX1416 and the RJ of Tn2009 in AP104 have been assigned GenBank accession no. DQ426906, DQ426907, and DQ426908, respectively.


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RESULTS AND DISCUSSION
 
Typing of isolates. Of the 16 erythromycin-resistant, mef(E)-positive isolates, 1 was also resistant to tetracycline and carried tet(M) besides mef(E) and 4 were resistant to tetracycline and to high levels of erythromycin and were found to carry mef(E), tet(M), and erm(B) (Table 2).


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  		TABLE 2. Properties of S. pneumoniae strains carrying mega or mega and other genetic elements

The isolates belonged to 10 different serotypes (Table 2). Three serotypes included multiple isolates: 9V (four isolates), 14 (three isolates), and 23F (two isolates). On the basis of the PFGE macrorestriction profiles, the isolates could be grouped into 11 genetically distinct PFGE types. Nine types included single isolates, while two types, type 1 and type 5, included the three serotype 14 isolates and the four serotype 9V isolates, respectively (Table 2).

Structure and insertion sites of mega. Mapping of mega revealed that both the mef(E) and msr(D) genes were present in all of the isolates. The overall size, structure, and sequences of the L and R ends of mega were similar to those previously described (9, 13). In seven isolates, the size of the amplicon of the intergenic region between mef(E) and msr(D) appeared approximately 100 bp smaller than that obtained with control strain PN150. This suggests that the intergenic region was truncated, as already described in mega elements found in North American isolates (13, 37).

A variety of different mega insertion sites were found. In tetracycline-susceptible isolates, five different sites could be recognized inside sequences annotated as open reading frames (ORFs) in the genome of R6 (GenBank accession no. NC_003098), three of which corresponded to already described insertion sites (13) and two of which were novel (Table 2). In one isolate (PT178), only the RJ of mega could be identified since the L end was flanked by a sequence showing no significant homology with pneumococcal or other bacterial genomes (Table 2). Sequence analysis of the mega insertion sites in comparison with the R6 genome showed a 2-bp deletion in the target sites, consisting of CA in three sites and CT or CG in one site each.

On the basis of previous results (10), linkage between mega and tet(M) was explored in the five tetracycline-resistant isolates but could be demonstrated in two isolates only, AP104 and PGX1416. Sequencing confirmed that in both isolates mega was inserted in orf6 of Tn916, the same site occupied by mega in Tn2009 (Fig. 2) (10).


Figure 2
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  		FIG. 2. Schematic representation of Tn2010, a novel Tn916 family transposon harboring mega and an erm(B) element. The black bar represents Tn916, and the white arrows indicate the locations of the relevant ORFs. The structures of the two inserted elements are boxed, and gray arrows indicate the ORFs. Black arrowheads indicate the positions of the primers used to amplify and sequence relevant portions of Tn2010 and its chromosomal junctions.

In the other three tetracycline-resistant isolates, all belonging to serotype 14, no linkage between mef(E) and tet(M) was demonstrated, since mega was found inserted in spr0166, which has been predicted to encode 3-methyladenine DNA glycosylase I in R6 (Table 2). This mega insertion site was the most common one in the isolates examined, being found in 7 out of 16 isolates. In these same isolates, mega showed a common structural feature, i.e., the intergenic-region deletion already described. Interestingly, in Streptococcus oralis we found a mega element with the same intergenic deletion that was inserted in a sequence similar to spr0166 (data not shown). The mega element lacks enzymes required for DNA transposition and is nonconjugative in pneumococci (9, 13). Evidence of common insertion sites in pneumococci and other streptococcal species supports the hypothesis that mega could spread by transformation among these species.

Composite elements. On the basis of previous investigations, the tetracycline-resistant isolates were examined by PCR mapping targeting Tn916-like elements (10).

In AP104, the amplicons obtained were of the same size as the corresponding fragments obtained with control strain PN150, confirming that AP104 harbors Tn2009.

In PGX1416, the amplicons were of the same size as the corresponding fragments obtained with control strain PN150, with the exception of the amplicon spanning the region from orf17 to orf20, which was larger than expected. With primer pairs EB2-O3 and O4-EB1, amplicons were produced indicating that erm(B) was located inside the orf17-orf20 fragment, oriented in the same direction as tet(M) and opposite to mef(E). Sequence analysis showed that erm(B) was contained in a fragment of 2,841 bp that included a transposase (tnp) (Fig. 2) and was integrated in orf20 of Tn916, at position 3,847 of the sequence of Flannagan et al. (GenBank accession no. U09422). This erm(B) element and the adjacent Tn916 regions showed identity to a sequence found in multidrug-resistant S. pneumoniae strains 670-6B (The Institute for Genomic Research, microbial genomes in progress at http://www.tigr.org) and G54 (GenBank accession no. AL449927) (11). In addition, the same erm(B) element was part of a transposon designated Tn916Erm (GenBank accession no. AY898750) found in Streptococcus cristatus, an oral streptococcal species.

The new composite element of approximately 26.3 kb that carries mef(E), tet(M), and erm(B) and is found in S. pneumoniae PGX1416 was designated Tn2010 (Fig. 2).

Tn2010 resembles previously described transposons with the addition of mega, such as Tn1545 described in S. pneumoniae by Caillaud and coworkers, which also includes the kanamycin resistance gene aphA-3 (3), or Tn3703, an S. pyogenes transposon derived from Tn916 by insertion of a 3-kb sequence carrying erm(B) (20).

Neither AP104, carrying Tn2009, nor PGX1416, carrying Tn2010, was able to transfer macrolide or tetracycline resistance by conjugation (transfer frequency, <10–9), confirming previous observations (10).

In the three isolates belonging to serotype 14 in which mega was not linked to tet(M), a linkage between tet(M) and erm(B) was found. PCR mapping and sequencing indicated the presence of another Tn916-like transposon, Tn3872, a composite element consisting of Tn917, carrying erm(B), inserted in orf9 of Tn916 (22).

Insertion sites of composite elements. Tn2009 of AP104 appeared to be inserted in a chromosomal site different from that previously described in PN150 and PN34 (10). By iPCR and sequencing, the LJ of Tn2009 in AP104 was found to be identical to the LJ of Tn5251 with Tn5252 (GenBank accession no. X90940). The R end was flanked by a sequence almost identical to a sequence of 670-6B. At the nucleotide level, the R end appeared to be integrated at nucleotide 114,074 of contig 2444 (The Institute for Genomic Research, microbial genomes in progress at http://www.tigr.org) at the same position where the L end of a Tn916-like transposon is integrated in 670-6B (Fig. 3). This complex context suggests that Tn2009 of AP104 might be part of a larger element.


Figure 3
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  		FIG. 3. Homology of the chromosomal LJ and RJ of composite transposons Tn2009 (A), Tn2010, and Tn3872 (B) with other pneumococcal sequences. The S. pneumoniae strains from which the sequences were derived are identified on the right. Transposons are represented by black lines. Boxes show magnified details of the junctions. Homologous regions are indicated by the same pattern. Black bars at the R end of Tn2009 and at the L end of Tn2010 and Tn3872 represent exogenous sequences showing no homology with other bacterial sequences. The insertion sites of Tn2009 in PN150 and PN34 have been described previously (10).

Analysis of the junctions of Tn2010 in the chromosome of PGX1416 revealed the presence of six nucleotides at the L end, likely representing the coupling sequence (4). The L end of Tn2010 appeared to be inserted in a region homologous to the R6 genome at base 1,743,232 (GenBank accession no. NC_003098), adjacent to spr1775. The R end appeared to be inserted at nucleotide 1,731,928 at the 3' end of spr1764. On the basis of the R6 genome, insertion of Tn2010 appeared to cause a deletion of approximately 11,300 bp. Both the L and R ends appeared to be flanked by brief sequence stretches (24 and 66 nucleotides, respectively) that did not show homology with the R6 genome, although the 66-bp sequence flanking the R end was identical to a sequence flanking the R end of a truncated Tn916-like transposon in G54 (Fig. 3) (11). The presence of exogenous sequences flanking the transposon's ends and of large chromosomal deletions occurring at the insertion site are features of Tn2010 already observed in Tn2009 (10).

Surprisingly, the insertion site of Tn3872 in the serotype 14 isolates was the same, at the nucleotide level, as that of Tn2010 in PGX1416 (Fig. 3). This and other similarities observed in the sequences flanking the Tn916-like transposons suggest the presence of preferential insertion sites for these elements in the pneumococcal chromosome.

Conclusions. Our findings confirm a tendency to the formation of modular aggregation of resistance determinants in mobile elements (36) that, in S. pneumoniae, appear to be centered on a core structure represented by Tn916. The mega element is one of the functional modules present in Tn2009 and Tn2010, and this characteristic differentiates it from the similar element Tn1207.1, which has not been found to associate with other resistance determinants in pneumococci. The Tn916 family of transposons shows a very broad host range, including more than 40 bacterial genera (30). This characteristic could contribute to the diffusion of Tn2009 and Tn2010 in pneumococci and other bacterial species. Tn2009 has been found in several pneumococcal isolates in Italy (5, 7) and recently has also been identified in a gram-negative species, Acinetobacter junii (26). Tn2010, which carries both erm(B) and mef(E), could be responsible for the spreading of the double macrolide resistance genes that have been recognized in particular pneumococcal clones in recent years (12, 19).


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ACKNOWLEDGMENTS
 
We thank Monica Monaco for typing the pneumococcal isolates.

This study was supported in part by grants from the Italian Ministero della Salute (Progetto "Sorveglianza della resistenza agli agenti antimicrobici" 2002 and Progetto Finalizzato 2003 "Controllo delle infezioni respiratorie") and from MIUR (FIRB 2005).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: (39) 0649902852. Fax: (39) 0649387112. E-mail: pantosti{at}iss.it. Back


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Antimicrobial Agents and Chemotherapy, October 2006, p. 3361-3366, Vol. 50, No. 10
0066-4804/06/$08.00+0     doi:10.1128/AAC.00277-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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