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Antimicrob Agents Chemother. Feb 2009; 53(2): 343–353.
Published online Nov 10, 2008. doi:  10.1128/AAC.00781-08
PMCID: PMC2630629

Genetic Elements Responsible for Erythromycin Resistance in Streptococci[down-pointing small open triangle]

During the last two decades, growing rates of erythromycin resistance have been reported in many countries among both Streptococcus pyogenes (29) and Streptococcus pneumoniae (63) clinical isolates. This trend was partly due to a further spread of the conventional methylase-mediated target site modification mechanism of resistance, but to an even greater extent it reflected the emergence of an active efflux-mediated mechanism of erythromycin resistance.

Besides less common mutations in 23S rRNA or ribosomal proteins, target site modification consists in posttranscriptional methylation of an adenine residue in 23S rRNA; this is caused by erm class gene-encoded methylases and usually results in coresistance to macrolide, lincosamide, and streptogramin B antibiotics (MLS phenotype) (62, 63). The traditional and widely predominant erm determinant in streptococci, erm(B), can be expressed either constitutively or inducibly and is usually associated with high-level resistance (62). A more recently described methylase gene, erm(TR) (92), an erm(A) subclass (84), is normally inducible (46, 55) and is widely distributed in S. pyogenes isolates (55, 58), whose resistance level appears to depend on the contribution of a drug efflux pump (51). erm(TR) has been detected in other beta-hemolytic Streptococcus species (59, 67, 110), whereas it is quite uncommon in S. pneumoniae (12, 34, 41, 43, 97, 106). erm(T), characterized by a very low G+C content (ca. 25%), was detected in inducibly erythromycin-resistant isolates of group D streptococci in Taiwan (99) and subsequently in the United States (40). Very recently, it has been detected in U.S. invasive isolates of inducibly resistant S. pyogenes that were negative for conventional erythromycin resistance determinants (111).

Efflux-mediated erythromycin resistance is associated, in streptococci, with a low-level resistance pattern affecting, among MLS antibiotics, only 14- and 15-membered macrolides (M phenotype) (96). Active efflux is encoded by mef-class genes, which include several variants. mef(A), the first mef gene to be discovered, was originally described in S. pyogenes (22) and was subsequently found to be widespread in this species, but it is also common in S. pneumoniae and other streptococcal species (60). mef(E), detected in S. pneumoniae shortly afterward (98), was found in a variety of other Streptococcus species (60), although it has only exceptionally been reported in S. pyogenes (4, 6, 87). Less common mef genes have been detected in S. pneumoniae [mef(I) (28)] and S. pyogenes [mef(O) (87)], and mef(B) and mef(G) new alleles have recently been described in group B (15) and group G (5, 15) beta-hemolytic streptococci, respectively. An msr class gene with homology to msr(A)—an ATP-binding cassette gene associated with macrolide efflux in Staphylococcus aureus (85)—is located immediately downstream of the mef gene. This msr gene is usually designated msr(D), even though different variants are associated with different mef genes. Studies carried out in pneumococci demonstrated that mef and msr(D) are cotranscribed, suggesting that the proteins encoded by the two genes may act as a dual efflux system (48), inducible by erythromycin (3). It has also been suggested that the msr(D)-encoded pump is capable of functioning independently of the one encoded by mef (3, 33).

Macrolide inactivation due to a phosphotransferase encoded by the mph(B) gene, formerly described only in gram-negative bacteria, has lately been detected in Streptococcus uberis, where the inactivation mechanism, however, conferred only resistance to spiramycin (1).

Until a decade ago, knowledge about the genetic elements responsible for erythromycin resistance in streptococci was virtually confined to a few plasmids or transposons carrying erm(B), then called ermAM or simply erm (56, 65). Such transposons mainly included Tn917, detected in Enterococcus faecalis when it was still regarded as a Streptococcus species (94, 101, 102), and Tn1545, detected in S. pneumoniae and also encoding resistance, besides tetracycline, to erythromycin and kanamycin (31, 32). Remarkably, Tn1545 was related to Tn916 (47), the prototype of a family of broad-host-range conjugative transposons conferring tetracycline resistance via the tet(M) gene (24, 81). Other Tn916-related erm(B)-carrying transposons early described in Streptococcus species (81) ceased to be reported in later studies. During the last decade, the discovery of the above-mentioned variety of erythromycin resistance genes in streptococci has been closely followed by the identification and characterization of a variety of genetic elements responsible for the resistance and its possible spread via intra- and interspecific transfer. Different erythromycin resistance genes are carried by different elements: in the case of mef genes, such close gene-element association was a major argument for recommending that mef(A), mef(E), and any future mef variants continue to be discriminated and kept apart (60) as opposed to being collected in a single class, mef(A), due to their high degree of similarity (84). This minireview is aimed at presenting such new knowledge about the genetic elements responsible for erythromycin resistance in streptococci. Elements and their essential characteristics are summarized in Table Table11.

TABLE 1.
Essential characteristics of established genetic elements responsible for erythromycin resistance in streptococci

erm(B)-CARRYING ELEMENTS

Tn917 and Tn3872.

Tn917 was originally identified on a nonconjugative plasmid from E. faecalis (101, 102). Exposure of cells to low concentrations of erythromycin stimulated its transposition to conjugative coresident plasmids, making it transferable to plasmid-free recipients of the same species (25, 102). Tn917 was sequenced in the mid-1980s (94); its subsequently updated sequence (5,614 bp) revealed five open reading frames (ORFs), of which orf2 is erm(B) and orf4 and orf5 are two specific transposition-related genes, tnpR (resolvase) and tnpA (transposase), respectively. After Tn917-like sequences had been demonstrated in erythromycin-resistant S. pneumoniae isolates (61), McDougal et al. (70) described in the same species a new composite structure, designated Tn3872 (23.6 kb), resulting from the insertion of Tn917 into orf9 of Tn916, at base 14525 of its published sequence (accession no. U09422), with the erm(B) gene of Tn917 thus linked to the tet(M) gene of Tn916. In recent studies of S. pyogenes (13), Tn3872-like elements were detected in several isolates, while in a few others Tn917 was probably carried by a plasmid. A composite element apparently corresponding to Tn3872 has been recently described in Streptococcus agalactiae (80). In mating experiments, Tn3872 could not be transferred from S. pneumoniae donors to S. pneumoniae (27, 70), S. pyogenes and E. faecalis (27) recipients, or from S. agalactiae to S. agalactiae (80). However, conjugal transfer did occur from S. pyogenes to S. pyogenes (13) and from Abiotrophia defectiva (Streptococcus defectivus) to E. faecalis (78), albeit at low frequencies.

Tn6002, Tn6003, Tn1545, and Tn2010.

An erm(B)/tet(M) linkage is also typical of a number of erm(B)-carrying elements (Tn6002, Tn6003, Tn1545, and Tn2010) that share with Tn3872 a Tn916-like structure, but where the erm(B)-carrying insertion is the so-called erm(B) element, a 2,847-bp fragment formed by five ORFs (orfP0 to orfP4), of which erm(B) is the third (orfP2). This fragment is found between orf20 (here detected in a larger form called orf20F, whose right end corresponds to orfP0) and orf19 of Tn916 at base 3847 of its published sequence. Insertion of the erm(B) element alone distinguishes transposon Tn6002 (20,880 bp), detected in both S. pyogenes (13) and S. pneumoniae (19, 27, 35) but originally found in Streptococcus cristatus and designated Tn916Erm, whose complete sequence (later updated) was available in GenBank since 2005 (accession no. AY898750). Intraspecific conjugal transfer of Tn6002 has been demonstrated in S. pyogenes (13), whereas transconjugants were obtained from a S. pneumoniae donor only using a S. pyogenes recipient (27). As shown by Cochetti et al. (27), another pneumococcal transposon, Tn6003 (25,101 bp), is distinguished by the further insertion into Tn6002 of the so-called MAS (for macrolide-aminoglycoside-streptothricin) element (4,225 bp). The MAS element is found within the erm(B) element between orfP0 and orfP1, at position 4110 of the Tn6002 sequence. A role in its insertion may have been played by a 222-bp sequence, absent in Tn916, detected both on the left end of the erm(B) element and on the right end of the MAS element (27). The MAS element appears to be a rearrangement of part of plasmid pRE25 (90) of E. faecalis and contains, from upstream to downstream, another erm(B) gene (lacking the stop codon) with its leader peptide; an aminoglycoside-streptothricin resistance cluster (aadE-sat4-aphA-3), with a 511-bp deletion in aadE; and an ORF identical to orf47 of pRE25 (27). Conjugal transfer of Tn6003 failed to S. pneumoniae and S. pyogenes recipients, whereas it was obtained to an enterococcal recipient (27). Tn6003 shares its resistance gene combination [erm(B) (MLS antibiotics), tet(M) (tetracyclines), and aphA-3 (kanamycin and structurally related aminoglycosides)] with Tn1545—originally detected in a multiply resistant clinical S. pneumoniae isolate—which was the first erm(B)-carrying element to be described in this species (31, 32). Since early studies, Tn1545 was shown to be transferable by conjugation to various gram-positive bacterial genera. In the years after its discovery, the genetic organization of Tn1545 has essentially been analyzed by restriction mapping (16); only specific portions have been sequenced, including its ends (17), the three above-mentioned resistance genes (18, 68, 103), and transposition-related genes such as int (integrase) and xis (excisase) (79), which are characteristically associated with the conjugative transposons of the Tn916 family (24, 81). The structural organization of Tn1545 has been elucidated quite recently, when two new regions of the transposon were sequenced and its genetic organization was compared with, and shown to be closely related to, that of Tn6003 (26). In particular, Tn1545 was found to contain a MAS element virtually identical (99.7% homology, accession no. AM903082) to that of Tn6003. Remarkably, the second erm(B) in the MAS element of Tn1545 lacked the stop codon, exactly like the one of Tn6003. The sole dissimilarity detected between the two transposons was a 1,245-bp insertion between orf13 and orf12 (at base 18833 of the Tn6003 sequence) found in Tn1545 (accession no. AM889142) but not in Tn6003, containing a putative IS1239 insertion sequence (26). This being the sole difference between Tn6003 (25,101 bp, as resulting from its complete sequencing) and Tn1545 (never completely sequenced), the actual size of the latter should be ~1 kb greater than the 25.3 kb reported originally on the basis of restriction mapping (16, 31, 32). Another erm(B)-carrying Tn916-like transposon (described in S. pneumoniae) is Tn2010 (26,390 bp, accession no. AB426620), which results from the insertion of a variant of the mef(E)-containing mega element (see below) into Tn6002 at position 17014 of the Tn6002 sequence and does not appear to be transferable by conjugation (35, 37). Less-common structures are also occasionally observed, such as two nonmobile Tn916-related elements with intermediate genetic organizations (perhaps vestiges of previous recombination events) lacking tet(M) but bearing both Tn917 and a complete or an incomplete MAS element (27).

Tn1116.

A special erm(B)-carrying transposon, Tn1116 (~50 kb), has been described in S. pyogenes by Brenciani et al. (13). It differs from the other Tn916-related elements by the involvement of Tn5397, a unique Tn916 family transposon originally found in Clostridium difficile (75, 83), where the tndX (resolvase) gene replaces int and xis. Tn1116 results from the insertion of erm(B)-containing DNA into the coding sequence of the tet(M) gene of Tn5397, at base 15019 of the published sequence of the transposon (accession no. AF333235); the whole portion of Tn5397 upstream of this insertion site is lacking. The erm(B)-containing insertion is largely unknown. However, the DNA region between the erm(B) and the tndX gene has been sequenced (accession no. AM411377), showing an insertion sequence, identical to the enterococcal IS1216 (64), between erm(B) and the truncated tet(M). Further sequencing in progress in our laboratory has shown homologies with the S. pyogenes plasmid pSM19035 (21). Tn1116 was easily transferred in intraspecific matings (13). A different Tn5397-related element, with a regularly expressed tet(M) instead of a truncated one, has been reported in S. pneumoniae isolates, but its actual features and organization have not yet been determined (19, 50).

Detection of and discrimination between erm(B)-carrying elements.

The maps of Tn916 and Tn916-related erm(B)-carrying transposons Tn3872, Tn6002, Tn6003, Tn1545, and Tn1116 are shown and compared in Fig. Fig.1.1. In order to detect and discriminate between these genetic elements, a PCR using the primer pair J12/J11 (the former targeting orf20 and the latter the intergenic region between orf18 and orf19 of Tn916) (27) is sufficient to distinguish among Tn3872, Tn6002, and Tn6003/Tn1545, which yield amplicons of 0.8, 3.6, and 7.9 kb, respectively (26). Tn6003 and Tn1545 can be distinguished using the primer pair O6/TETM2dg [the former targeting orf13 and the latter tet(M) of Tn916], that yield amplicons of 2.4 and 3.6 kb, respectively (26). This scheme does not consider Tn2010, distinguished by the presence of the mef(E) gene. Transposon Tn1116 can be detected by pairing primers ERMB2 and tndX-2 (13), targeting erm(B) and tndX, respectively, and yielding a PCR product of 6.5 kb.

FIG. 1.
erm(B)-carrying elements. The erm(B) gene is indicated as a checkered red arrow. Light-blue arrows indicate Tn916 and Tn916-related ORFs other than tet(M) (blue). Pink, green, and orange arrows indicate ORFs from Tn917, the erm(B) element, and the MAS ...

Spread and prevalence of erm(B)-carrying elements.

Very recently, some data have become available about the spread and prevalence of the different erm(B)-carrying elements in streptococcal populations. In S. pneumoniae isolates, the erm(B) gene seems to be carried primarily by Tn917 or Tn917-containing elements in Japan (76), whereas Tn6002 ranks first (half to two-thirds) in Italy (26, 27) and Spain (19), followed by Tn3872 and, far distanced, by Tn6003/Tn1545. Interestingly, in the only study distinguishing between Tn6003 and Tn1545, only the former was detected (26). In fact, most epidemiological surveys of erythromycin-resistant pneumococci have neglected the phenotypic or genotypic assessment of kanamycin susceptibility; this may have resulted in some confusion not only between Tn1545 and Tn6003 but also between Tn1545 and Tn6002. However, the few studies addressing the question confirm that kanamycin resistance is associated with a tiny minority of erm(B)-positive pneumococci (19, 49, 57, 70, 93). It is noteworthy that Tn6003 and the MAS element have thus far been demonstrated only in S. pneumoniae (27), whereas there is as yet no evidence of their presence in erm(B)-carrying isolates of S. pyogenes (13). Conversely, Tn1116 has been detected only in the latter species: it was invariably associated with erm(B)-positive isolates with the inducible resistance phenotype, while a greater variability has been recorded in those with the constitutive phenotype, where Tn6002 accounted for almost half and Tn3872 for more than one-quarter of the isolates (13).

erm(TR)-CARRYING ELEMENTS

In S. pyogenes, the erm(TR) gene, formerly reported as prophage associated in a serotype M6 isolate (10), has subsequently been reported to be carried on an integrated conjugative element (ICE) in a serotype M4 strain whose genome was completely sequenced (accession no. CP000262) (11). ICEs are large segments of exogenous DNA with the characteristics of conjugative transposons or plasmids; they are regarded as putatively mobile even when their transferability has not been shown experimentally (11). In particular, erm(TR) was found in a 49-kb ICE, designated ICE 10750-RD.2, that is integrated into the hsdM chromosomal gene encoding host DNA restriction modification methyltransferase. Remarkably, other antibiotic resistance genes have been detected in the erm(TR)-flanking region, both upstream (tetronasin) and downstream (spectinomycin) of erm(TR).

The erm(TR) gene is uncommon in S. pneumoniae but has been shown to be carried on a genetic element, designated Tn1806, possibly representing a transposon/prophage remnant chimera (20). Tn1806 (56 kb, i.e., ca. 7 kb larger than ICE 10750-RD.2 from S. pyogenes) is inserted into a chromosomal ORF homologous to spr0790 of S. pneumoniae R6, corresponding to an hsdM gene coding for a type I restriction modification system. Sequencing of an ~11-kb erm(TR)-containing DNA fragment from Tn1806 and of its left and right junctions (accession no. EF469826) disclosed substantial homologies to ICE 10750-RD.2, despite the identification of ORFs unique to either element. Tn1806 did not appear to be conjugative but could be transferred by transformation to a pneumococcal recipient (20).

Before the detection and characterization of ICE 10750-RD.2 from S. pyogenes and of Tn1806 from S. pneumoniae, mating experiments had demonstrated the transfer of the erm(TR) gene, contained in putative elements of 30 to 40 kb, from S. pyogenes to the same and to other gram-positive species (54). Sequence analysis of the transposable element from one of these donors (accession no. FM162351) demonstrated extensive similarities to ICE 10750-RD.2, but to an even greater extent to Tn1806. The sequences of the erm(TR)-flanking regions from the three elements are compared in Fig. Fig.22.

FIG. 2.
Schematic and comparative representation of the erm(TR)-flanking regions from three erm(TR)-carrying elements (Tn1806 from S. pneumoniae, and an unnamed element [sequence accession no. FM162351] and ICE 10750-RD.2 from S. pyogenes). The erm(TR) gene is ...

erm(T)-CARRYING ELEMENTS

The genetic organization of the erm(T) gene and its flanking regions was described in group D streptococci (S. gallolyticus subsp. pasteurianus) (104). The erm(T)-carrying genetic element has a chromosomal location, but only a ca. 4.1-kb fragment has been sequenced and examined, revealing that erm(T) was flanked by two IS1216V-like insertion sequences with the same polarity.

A plasmid-borne erm(T) has lately been described in S. pyogenes (111). The plasmid, designated pRW35 (4,962 bp), contains three distinct ORFs, erm(T) and two ORFs highly similar to plasmid replication and transfer genes found in broad-host-range plasmids, a finding consistent with the hypothesis that pRW35 is capable of broad dissemination (111).

mef(A)-CARRYING ELEMENTS

The first mef(A)-carrying structure to be discovered was Tn1207.1 (7,244 bp), a chromosomal element described in S. pneumoniae by Santagati et al. (89) and considered as a defective transposon because the 5′ end of orf8 appears truncated. It contains eight ORFs, of which mef(A) and msr(D) are the fourth and the fifth; while orf2 is homologous to site-specific recombinases of gram-positive bacteria, orf6, orf7, and orf8 are homologous to three ORFs of the pneumococcal conjugative transposon Tn5252 (2). Tn1207.1 is integrated at a specific site of the pneumococcal chromosome into celB, a late competence gene (notably, Tn1207.1-carrying pneumococci are unable to become competent [36]). Tn1207.1 has been reported to be transferable by transformation but not by conjugation (89), even though variable results have been obtained in mating experiments with different pneumococcal donors (36).

Whereas Tn1207.1 is the sole recognized element carrying mef(A) in S. pneumoniae, in S. pyogenes it is not detected as such, but as part of larger composite elements, all shown to be chimeric, i.e., resulting from insertion of a transposon (identical or related to Tn1207.1) into a prophage (9, 53). In tetracycline-susceptible M-phenotype isolates of this species, a regular Tn1207.1 (with a nontruncated orf8) forms the left end of Tn1207.3 (52,491 bp) (88) or is part of Φ10394.4 (58,761 bp) (9, 10). Tn1207.3 and Φ10394.4 are closely related elements: they are integrated into the same chromosomal gene (comEC) and are inserted into the same prophage, as initially suggested by PCR assays (14) and subsequently confirmed by sequence analysis of the large fragments of phage origin located to the right of Tn1207.1 (accession no. AY657002). Φ10394.4 differs from Tn1207.3 by an additional left-hand region that has been reported to be ~6-kb long (with one ORF encoding an R28-like protein) but also to be quite variable in size (9), suggesting that Tn1207.3 could represent one extreme, completely lacking this region, of the variability range (53). Tn1207.3, which appears to be more common than Φ10394.4 in mef(A)-positive, tetracycline-susceptible S. pyogenes isolates (14, 39, 45), has also been occasionally detected in other beta-hemolytic Streptococcus species (45, 67). Tn1207.3 has been shown to be transferable in intra- and interspecific mating experiments (88). In tetracycline-resistant M-phenotype isolates of S. pyogenes, where tetracycline resistance is consistently mediated by the tet(O) determinant (52), mef(A) is linked to tet(O) in a mobile element, also chimeric, where, however, the prophage is different from the one shared by Tn1207.3 and Φ10394.4 (53). In fact, there is a variety of related tet(O)-mef(A) elements in which mef(A), located 2.3 to 5.5 kb downstream of tet(O), is harbored by a range of changeable and defective variants of Tn1207.1: of the eight typical ORFs of the latter, only msr(D) and a modified orf6, in addition to mef(A), were detected by PCR in all isolates tested, while orf1 and orf2 were always undetectable, and orf3, orf7, and orf8 were found in variable percentages (14). The one currently designated Φm46.1 (~60 kb), ostensibly the most common of such elements, has been shown to be transferable in mating experiments to S. pyogenes, but not to an enterococcal recipient (52), and is not integrated within the comEC gene (14). An ~12.1-kb region of Φm46.1, from upstream of the tet(O) gene through the end of a Tn1207.1-related transposon, has been sequenced (accession no. AJ715499), disclosing four new ORFs (orfA to orfD) downstream of the tet(O) gene; tet(O) was 5,717 bp upstream of mef(A) (14). It should be noted that tetracycline-susceptible M-phenotype isolates of S. pyogenes are usually SmaI nontypeable by pulsed-field gel electrophoresis (82, 105) because their DNA is made refractory to SmaI restriction by a DNA-modifying methyltransferase (45). This enzyme is encoded by a restriction modification cassette located just downstream of Tn1207.1 in the conserved region of phage origin of Tn1207.3 and Φ10394.4 (42). The cassette is absent in Φm46.1, a finding consistent with the SmaI typeability of tetracycline-resistant M-phenotype isolates (7).

The maps of Tn1207.1, Tn1207.3, Φ10394.4, and Φm46.1 are represented and compared in Fig. Fig.33.

FIG. 3.
mef(A)-carrying elements. Yellow arrows indicate Tn1207.1 and related ORFs other than mef(A) (oblique pink stripes) and msr(D) (horizontal orange stripes). A dark blue arrow indicates tet(O). Gray areas between ORF maps indicate areas of homology.

mef(E)-CARRYING ELEMENTS

The genetic structure typically carrying the mef(E) gene in S. pneumoniae, first described by Gay and Stephens (48), has been designated as mega (macrolide efflux genetic assembly) element. It contains five ORFs having related sequences to the last five ORFs of Tn1207.1, of which mef(E) [91.6% homology to the mef(A) gene of Tn1207.1] is the first and msr(D), originally named mel, is the second. The mega element has a variety of insertion sites in the pneumococcal chromosome, possibly associated with different variants. The deposited mega sequence (5,532 bp) refers to the first investigated variant (48); this is probably less common than a slightly smaller variant, with a 99-bp deletion in the intergenic region between mef(E) and msr(D), detected in pneumococci isolated in North America (48, 109) and Italy (35). The mega element was shown to be transferable by transformation (36, 48) and not by conjugation (36). The mef(E) gene was detected in other streptococcal species, but the carrier element has been investigated only in Streptococcus salivarius. In this species, Stadler and Teuber (95) described a new mega variant (5,511 bp) whose sequence was almost identical to that of the pneumococcal element; an additional ORF (orf6) was detected between orf4 and orf5, both partly modified compared to the corresponding ORFs in the pneumococcus. The mega element from S. salivarius was transformed into a pneumococcal recipient (95). A mega element 100% identical to the one of S. salivarius, but with an additional ORF (orf7) located upstream of mef(E), was detected in S. pneumoniae in the composite transposons Tn2009 (23.5 kb) (38) and Tn2010 (26,390 bp) (35, 37). The former results from insertion of the mega variant in orf6 of transposon Tn916, at position 14166 of its published sequence, the latter from the same insertion at the same position in Tn6002. In both Tn2009 and Tn2010 mef(E) is oriented opposite to tet(M) of Tn916. Tn2009 was shown to be transferable by transformation (38). Neither Tn2009 nor Tn2010 could be transferred by conjugation to pneumococcal or enterococcal recipients (35). The maps of the mef(E)-carrying elements are shown and compared in Fig. Fig.44.

FIG. 4.
mef(E)-carrying elements. Yellow arrows indicate Tn1207.1-related ORFs other than mef(E) (oblique pink stripes) and msr(D) (horizontal orange stripes). A blue arrow indicates tet(M). Green arrows indicate ORFs from the erm(B) element other than erm(B) ...

ELEMENTS CARRYING OTHER mef GENES

Of the mef genes other than mef(A) and mef(E) described thus far (5, 15, 28, 87), the gene-carrying element has been studied only for mef(I), detected in S. pneumoniae and exhibiting 91.4 and 93.6% homologies to the mef(A) gene of Tn1207.1 and the mef(E) gene of the mega element, respectively (28). As shown by Mingoia et al. (73), mef(I) is carried by an apparently nonmobile composite structure, designated 5216IQ complex (30,505 bp), consisting of two halves of comparable size (Fig. (Fig.5).5). The left half (15,316 bp) is formed by parts of the known transposons Tn5252 (2) and Tn916, the latter containing a silent tet(M) gene; the Tn916 ORFs upstream of tet(M) are lacking, as is most of Tn5252. The right half (15,115 bp) of the 5216IQ complex is formed by a new fragment, designated the IQ element, that contains the mef(I) gene with a new adjacent msr(D) gene variant, in the context of a genetic organization totally unlike those of the genetic elements carrying other mef genes. In the IQ element, mef(I) and msr(D) are the seventh and the eighth of 13 ORFs, of which the first and the last are two identical transposase genes and the twelfth is a catQ chloramphenicol acetyltransferase determinant. This was the first time this resistance gene, thus far detected only in Clostridium perfringens (91), was demonstrated in S. pneumoniae and shown to be linked with a mef gene. The IQ element should probably be considered as a novel transposon, although it does not appear to be mobile in the genetic context of the pneumococcus. The right junction of the 5216IQ complex was just upstream of the spr0601 gene and the left junction just downstream of the spr1199 gene of the S. pneumoniae R6 chromosome. However, further sequencing beyond the left junction disclosed a chromosomal organization more similar to that of the virulent pneumococcus TIGR4 (100) than to that of strain R6.

FIG. 5.
mef(I)-carrying element (5216IQ complex). Purple arrows indicate ORFs from transposon Tn5252. Light-blue arrows indicate ORFs from transposon Tn916 other than tet(M) (blue). White arrows indicate ORFs from the IQ element other than mef(I) (oblique pink ...

CONSIDERATIONS AND CONCLUSIONS

The streptococcal genetic elements considered here, chosen and reviewed as carriers of macrolide resistance determinants (erm or mef/msr class genes), also carry a number of other genes conferring resistance to a variety of other antibiotics, including tetracyclines, aminoglycosides, spectinomycin, and chloramphenicol, but also minor drugs such as streptothricin and tetronasin. On the other hand, recruitment of exogenous resistance genes and their concentration in particular genetic elements, as exemplified in gram-negative bacteria by the integrons and superintegrons (69), is the most rapid adaptation mechanism against antimicrobial compounds and is likely to have an important role in the horizontal gene transfer and the evolution of bacterial genomes (86). Del Grosso et al. (38) advocated that, in gram-positive bacteria such as the streptococci, a similar function can be exerted by conjugative transposons such as Tn916-like elements, which are highly evolved for broad-host-range transfer and highly capable of capturing other resistance elements to form composite structures, thus playing a major role in disseminating multiple drug resistances (24, 81). In some instances, particularly when the species S. pyogenes is involved, a major role can also be played by bacteriophages (8).

Most erm(B)-carrying elements detected in streptococci result from the insertion of erm(B)-containing DNA into conjugative transposons of the Tn916 family. The tet(M) gene is typically carried by these elements (24, 81), and until a short time ago this steady tet(M)-Tn916 association was held to be consistent with the fact that tetracycline-resistant erm(B)-positive streptococci are far more prevalent than tetracycline-susceptible ones. However, recent studies have unexpectedly demonstrated that tetracycline-susceptible erm(B)-positive isolates of both S. pyogenes (13, 74) and S. pneumoniae (27, 74) also most often carry Tn916-related elements, where, however, the tet(M) gene is present in a silent form. Retrospectively, it should be noted that tetracycline-susceptible tet(M)-positive isolates had been observed in some collections of pneumococci (4, 30, 66, 70, 77, 107).

Conjugal transfer of transposons or other genetic elements is often efficient from S. pyogenes but is lacking (or inefficient) from S. pneumoniae. Indeed, the latter has highly developed transformation systems, which are the basis for the unique recombination-mediated genetic plasticity that is a distinctive feature of the species (23). Conversely, natural transformation is not a common event in S. pyogenes, although the species has many of the genes essential for competence and transformation. It has been suggested that, in S. pyogenes strains, competence may have been lost due to the growing role assumed by phages (44); in fact, S. pyogenes is the only Streptococcus species containing complete bacteriophage genomes, indicating that this mechanism of horizontal gene transfer is an important factor in gene acquisition and/or loss, strain heterogeneity, and the overall evolution of the species (8, 44). In particular, a chimeric nature—i.e., a transposon inserted into a prophage—has been demonstrated for all recognized mef(A)-carrying elements of S. pyogenes, i.e., the closely related Φ10394.4 (9, 10) and Tn1207.3 (88) (inserted into the same prophage) in tetracycline-susceptible isolates, and the tet(O)-mef(A) elements (e.g., Φm46.1, inserted into a different prophage) in tetracycline-resistant isolates (53). Both in the case of mef(A), primarily carried by a nonconjugative transposon such as Tn1207.1 (89), and in the case of tet(O), believed to be unable to move from one chromosome to another prior to the discovery of the tet(O)-mef(A) elements (14, 52), a nontransferable resistance determinant of S. pneumoniae (or of other streptococci from the upper respiratory tract) could have become easily transferable once transfer to S. pyogenes and integration into the prophage had successfully occurred. In the 1970s, several experimental studies provided evidence that prophages might have participated in the dissemination of erythromycin resistance among S. pyogenes isolates (8, 72). The subsequent hypothesis that the resistance determinant involved in those transduction experiments was mef(A) (8) appears far sounder now that specific prophage-associated mef(A) elements have been identified. Although the molecular mechanisms mediating the horizontal transfer of mef(A) have not yet been clearly defined, several lines of evidence suggest that transduction can play a role (9, 72). In any case, phage transfer is likely to play a critical role in the emergence of active efflux as the most widespread mechanism of macrolide resistance in this species.

Acknowledgments

We thank heartily Alessandro Bacciaglia, Andrea Brenciani, Ileana Cochetti, Marina Mingoia, and Emily Tili for their invaluable collaboration and helpful discussions.

Footnotes

[down-pointing small open triangle]Published ahead of print on 10 November 2008.

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