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Antimicrobial Agents and Chemotherapy, September 2000, p. 2585-2587, Vol. 44, No. 9
Dipartimento di Biologia Molecolare, Sezione
di Microbiologia, Università di Siena, 53100 Siena,1 and Dipartimento di Scienze
Microbiologiche, Università di Catania,
Catania,2 Italy
Received 14 February 2000/Returned for modification 21 April
2000/Accepted 27 June 2000
The mef(A) gene from a clinical isolate of
Streptococcus pneumoniae exhibiting the M-type resistance
to macrolides was found to be part of the 7,244-bp chromosomal
element Tn1207.1, which contained 8 open reading frames.
orf2 encodes a resolvase/invertase, and orf5 is
a homolog of the macrolide-streptogramin B resistance gene
msr(SA).
A new mechanism of resistance to
macrolides based on an efflux system has recently emerged among
clinical isolates of Streptococcus pyogenes and
Streptococcus pneumoniae (35, 37). It is due to
the presence of macrolide efflux (mef) genes conferring the M phenotype, which is characterized by resistance to macrolides and
sensitivity to lincosamides and streptogramin B antibiotics (37). mef(A), originally described in
S. pyogenes (8), and mef(E),
originally described in S. pneumoniae (38), are
90% identical and were assigned to the same mef(A) class of
macrolide resistance determinants (32). Genes of the
mef(A) class (i) were found in different gram-positive
genera including Corynebacterium, Enterococcus,
Micrococcus, and Streptococcus, (ii) were never found associated with extrachromosomal plasmid DNA, and (iii) in some
instances were shown to be transferred by conjugation (3-5, 10,
14, 15, 17, 26, 40). Both mef(E) and mef(A) have been reported to occur in pneumococci from Italy (19, 20, 25). Here we report the first characterization of a
mef-carrying genetic element. The recently proposed
nomenclature for macrolide resistance determinants (32)
was adopted throughout this paper.
Transfer of mef(A).
To isolate and characterize
the genetic element that carries the macrolide efflux gene of S. pneumoniae STR174b, mef(A) was transferred from STR174b
to Rx1 (34), a rough (unencapsulated) laboratory strain of
pneumococcus. Clinical strain STR174b, isolated from sputum and
exhibiting the M-type resistance to macrolides, was used as donor in
conjugation and transformation experiments (Table
1). The mef(A) gene could not
be transferred from STR174b by conjugation (<3.4 × 10
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of a Genetic Element Carrying the
Macrolide Efflux Gene mef(A) in Streptococcus
pneumoniae
ABSTRACT
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8 transconjugants per donor), in filter matings
performed as described by Smith and Guild (36), while
successful transfer to Rx1 was accomplished by transformation (Table
1). Competent cells of Rx1 were prepared as previously described
(28); transforming DNA of STR174b was added at 1 µg per ml
of competent cells, and erythromycin-resistant transformants were
isolated at a frequency of 4.7 × 104 per recipient
(Table 1). MF4, a representative transformant, was kept for further
analysis. MF4 showed the same resistance phenotype as the parent
clinical strain, being resistant to erythromycin (MIC, 8 µg/ml) and
sensitive to lincosamides and streptogramin B antibiotics. Again,
mef(A) could not be transferred by conjugation in matings
where MF4 was the donor and S. pneumoniae, S. pyogenes, and Streptococcus gordonii were used as
recipients (Table 1).
TABLE 1.
Transfer of Tn1207.1
Sequencing and sequence analysis. The nucleotide sequence of the DNA adjacent to mef(A) was determined in both STR174b and MF4, using templates obtained by ligation-mediated and/or inverse PCR (24, 31). Direct automated sequencing of long PCR fragments was performed as previously described (11, 12). The software BLAST (2) was used to conduct homology searches of the GenBank database and the microbial genome databases available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) and at the WIT Computational Biology Group at Argonne National Laboratory website (http://wit.mcs.anl.gov./WIT2/CGI). Sequence analysis was carried out at the Baylor College of Medicine Search Launcher server (http://dot.imgen.bcm.tmc.edu:9331/index.htlm). RNA structure prediction was done using RNA structure 2.5 (21).
Nucleotide sequence of the mef(A) element.
Both in STR174b and in MF4 the mef gene was shown to
be 100% identical to the already described mef(A) gene
(8) and was found to be part of a chromosomal element which
was designated Tn1207.1. Tn1207.1 was found to be
7,244 bp in size (Fig. 1), and its
nucleotide sequence was identical in both strains. Sequence data
analysis showed the presence of 8 open reading frames
(ORFs), of which the first 5 have the same direction of
transcription, while orf6, orf7, and
orf8 are oriented opposite to the others (Fig. 1). Between
orf3 and mef(A) there is an intergenic region with a high potential for the formation of hairpins (21).
Palindromes indicative of terminators can be detected downstream of
orf1 and orf3. Terminal repeats could not be
detected.
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Chromosomal insertion site. Tn1207.1 was found integrated at a specific site in the chromosome of S. pneumoniae, downstream of the A nucleotide in position 1676 of the celB gene (27). Both in clinical isolate STR174b and in transformant MF4, the integration of Tn1207.1 caused a 1,947-bp deletion in the pneumococcal genome (nucleotides 1393 to 3339 of contig 4139) (The Institute for Genomics Research [http://www.tigr.org]) involving the 3' end of celB (nucleotides 2775 to 3339 of contig 4139) and the 5' end of orf436 (nucleotides 1393 to 1784 of contig 4139) (WIT website [see above]).
ORFs of Tn1207.1.
To identify genes homologous to the
ORFs of Tn1207.1, homology searches were done at a protein
level, using the BLAST software (2). The E value, which
is considered a very reliable statistical scoring measure
(7), is reported for each homolog detected by the search
(Table 2). No homolog was found for
orf1 and orf3, while orf2 was found
to be homologous to site-specific recombinases of genetic elements
of gram-positive bacteria. These elements included
chromosomal elements of Staphylococcus aureus
(13) and Bacillus subtilis (39), a
bacteriophage of Listeria monocytogenes (16), and
a transposon of Clostridium perfringens (6)
(Table 2). An additional ORF potentially involved in determining
resistance to macrolides and streptogramin antibiotics was found
adjacent to mef(A). orf5, whose coding sequence
starts 119-bp downstream of the mef(A) stop codon (Fig. 1),
was found to be homologous to msr(SA) (22) and
vga(A) (1). msr(SA) confers resistance to macrolides and streptogramin B, and vga(A) confers
resistance to streptogramin A antibiotics. Both genes are
putative members of the ABC transporter superfamily and mediate
resistance by encoding an antibiotic-specific efflux pump
(32). orf6, orf7, and orf8 are homologous to 3 ORFs of the pneumococcal conjugative transposon Tn5252 (23). The 5' end of orf8
appears truncated since its gene product is homologous to the
C-terminal portion of UmuC, an UV-resistance protein encoded by
Tn5252 (Table 2). No function is known for the gene products
of orf6 and orf7 and the Tn5252 homologs. Even if no significant homology can be detected at the nucleotide level, orf6 to orf8 and their
Tn5252 homologs show a similar organization characterized by
overlapping ORFs. orf6 and orf7 overlapped by 14 bp, and orf7 and orf8 overlapped by 4 bp.
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Conclusions. In this work we characterized a genetic element that carries the macrolide efflux gene mef(A) in a clinical isolate of S. pneumoniae exhibiting the M-type resistance to macrolides. mef(A) was found to be part of a chromosomal genetic element designated Tn1207.1. Tn1207.1 should be considered a defective transposon, since it terminates with a truncated ORF at the right side, while in different clinical isolates it is integrated at the same chromosomal site and has the same overall genetic organization but is larger in size at its right side (Santagati and colleagues, unpublished data). The presence of an ORF potentially encoding a site-specific recombinase of the resolvase/invertase family resembles clostridial transposons Tn4451, Tn4453, and Tn5397, in which the TnpX and TndX resolvases are involved in the transposon excision process (6, 9, 18, 41). Although it has been reported that mef genes from S. pneumoniae and group C streptococci can be transferred by conjugation (15, 17), Tn1207.1 appeared to be nonconjugative in nature.
Nucleotide sequence accession number. The complete nucleotide sequence of Tn1207.1 was assigned GenBank accession no. AF227520.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants from CNR (P. F. Biotecnologie) and MURST (Cofinanziamento 1999).
We thank Alessandra Zanchi for help in the initial phases of this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbiologia, Università di Siena, Via Laterina 8, 53100 Siena, Italy. Phone: 39-0577-233430. Fax: 39-0577-233870. E-mail: pozzi{at}unisi.it.
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