Susceptibilities to macrolides were evaluated in 267 Streptococcus pneumoniae isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried mef. Strains that carried both mef(A) and mef(E) were found: 17 strains carried mef(A) and 3 carried mef(E). The characteristics of the strains carrying the mef genes and the properties of the mef-containing elements were studied. Strains carrying mef(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three mef(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the mef-containing element, which encompassed mef and the msr(A) homolog, were identical among the three mef(E)-positive strains and among the three mef(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all mef(A)-positive strains, the mef element was inserted in celB, which led to impairment of the competence of the strains. In line with insertion of the mef(E) element at a different site, the competence of the mef(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three mef(A) strains but from none of three mef(E) strains. Due to the important different characteristics of the strains carrying mef(A) or mef(E), we suggest that the distinction between the two genes be maintained.Macrolide resistance in Streptococcus pneumoniae is typically due to acquisition of the erm(B) gene, which mediates ribosomal modification (10), or the mef gene, which encodes a drug efflux pump (28). Recently, mutations in the 23S rRNA or ribosomal proteins of S. pneumoniae have been found to confer erythromycin (ERY) resistance in some clinical isolates (30).The Mef pump confers a low to moderate level of resistance to 14-and 15-membered macrolides but not to lincosamide or streptogramin B antibiotics (M phenotype). Of the two variants of the mef gene, mef(A) was originally found in Streptococcus pyogenes (3) and mef(E) was originally found in S. pneumoniae (29). mef(A) and mef(E) are 90% identical at the nucleotide level and were assigned to the same class of macrolide resistance determinants (22). In most subsequent studies, mef was detected by a PCR assay that did not distinguish between the two variants (27). However, the two variants were considered species specific; therefore, if a mef gene was found in S. pneumoniae, it was generally assumed to be mef(E) (9, 16, 26). However, mef(A) was shown to be present in macrolideresistant Italian isolates of S. pneumoniae (18).Genetic elements carrying mef genes in S. pneumoniae were recently detected and characterized. The mef(A)-carrying element is a 7.2-kb defective transposon (Tn1207.1) that contains eight open reading fra...

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The association between the macrolide efflux gene mef(E) and the tet(M) gene was studied in two clinical strains of Streptococcus pneumoniae that belonged to serotypes 19F and 6A, respectively, and that were resistant to both tetracycline and erythromycin. The mef(E)-carrying element mega (macrolide efflux genetic assembly; 5,511 bp) was found to be inserted into a Tn916-like genetic element present in the chromosomes of the two pneumococcal strains. In both strains, mega was integrated at the same site, an open reading frame identical to orf6 of Tn916. The new composite element, Tn2009, was about 23.5 kb and, with the exception of the tet(M)-coding sequence, appeared to be identical in both strains. By sequencing of the junction fragments of Tn2009 at the site of insertion into the chromosome, it was possible to show that (i) the insertion site was identical in the two clinical strains and (ii) the integration of Tn2009 caused a 9.5 kb-deletion in the pneumococcal chromosome. It was not possible to detect the conjugal transfer of Tn2009 to a recipient pneumococcal strain; however, transfer of the whole element by transformation was shown to occur. It is possible to hypothesize that Tn2009 relies on transformation for its spread among clinical strains of S. pneumoniae.

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.

The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan19F-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.

Zinc metalloproteinase genes in clinical isolates of Streptococcus pneumoniae: association of the full array with a clonal cluster comprising serotypes 8 and 11A Pneumococci display large zinc metalloproteinases on the surface, including the IgA protease, which cleaves human IgA1 in the hinge region, the ZmpC proteinase, which cleaves human matrix metalloproteinase 9 (MMP-9), and two other proteinases, ZmpB and ZmpD, whose substrates have not yet been identified. Surface metalloproteinases are antigenic and have been linked to virulence. The genes encoding these proteinases reside in three distinct loci: two loci specific for zmpB and zmpC, and a third, the iga locus, containing iga and zmpD. Data obtained by this and other groups have shown that pneumococcal metalloproteinase genes are transcribed and yield mature and enzymatically active proteins. Since the presence of the four proteinase genes is variable in the pneumococcal strains whose genomes have been sequenced, the presence of these genes in a collection of 218 pneumococcal isolates, mostly from invasive disease, was investigated. The data showed that zmpB and iga were present in all the isolates examined, while zmpC and zmpD were present in a variable proportion of the isolates (in 18 and 49 %, respectively). Interestingly, isolates carrying both zmpC and zmpD were found to belong mainly to two serotypes (sts), 8 and 11A. By molecular typing, st 8 and st 11A isolates appeared to belong to the same clonal cluster. The presence of these two additional metalloproteinases could contribute to the fitness of particular pneumococcal clones. INTRODUCTIONStreptococcus pneumoniae (pneumococcus) is a common and important human pathogen. Pneumococci are the chief cause of community-acquired pneumonia and bacterial meningitis, the second-most-prevalent cause of sepsis, and one of the main causes of otitis media (Gillespie, 1989). The main virulence factors of S. pneumoniae are the capsule, the pneumolysin that is a cholesterol-dependent cytolysin, and some pneumococcal surface proteins and enzymes, including PspA, PspC, neuraminidase and hyaluronidase (Paton et al., 1993;Mitchell, 2000;Chapuy-Regaud et al., 2003;Iannelli et al., 2004). A characteristic feature of S. pneumoniae is the presence of large zinc metalloproteinases on the cell surface. These enzymes have a common structure (Pfam Accession no. PF02395) and are present not only in S. pneumoniae but also in related oral streptococci, such as Streptococcus sanguis, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis .From the published genome sequences, it can be inferred that S. pneumoniae possesses two (strain R6, rough derivative of serotype 2; Hoskins et al., 2001), three (strain TIGR4, serotype 4; Tettelin et al., 2001) or even four (strain G54, serotype 19F;Dopazo et al., 2001) zinc metalloproteinase genes, depending on the strain. All the above-mentioned strains possess iga and zmpB, encoding the IgA protease and ZmpB, respectively. In addition, strains TIGR4 and G54 possess the metal...

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