Abstract
In Streptococcus pyogenes, efflux-mediated erythromycin resistance is associated with the mef gene, represented mostly by mef(A), although a small portion of strains carry different mef subclasses. We characterized the composite genetic elements, including mef subclasses other than mef(A), associated with other resistance genes in S. pyogenes isolates. Determination of the genetic elements was performed by PCR mapping. The strains carrying mosaic mef(A/E), in which the 5′ region was identical to mef(A) and the 3′ region was identical to mef(E), also carried tet(O). The two genes were found enclosed in an element similar to S. pyogenes prophage Φm46.1, designated the Φm46.1-like element. In S. pyogenes strains carrying mef(E) and tet(M), mef(E) was included in a typical mega element, and in some strains, it was physically associated with tet(M) in the composite element Tn2009. S. pyogenes strains carrying mef(I) also carried catQ; the two genes were linked in a fragment representing a portion of the 5216IQ complex of Streptococcus pneumoniae, designated the defective IQ element. In the only isolate carrying a novel mef gene, this was associated with catQ and tet(M) in a genetic element similar to the 5216IQ complex of S. pneumoniae (5216IQ-like complex), suggesting that the novel mef is in fact a variant of mef(I). This study demonstrates that the composite elements containing mef are shared between S. pyogenes and S. pneumoniae and suggests that it is important to distinguish the mef subclass on the basis of the genetic element containing it.
INTRODUCTION
Efflux-mediated macrolide resistance in Streptococcus pyogenes and Streptococcus pneumoniae is conferred by mef and the adjacent msr(D) gene, which code for a two-component transport system (1). The resistance phenotype acquired by the microorganisms is defined as M, since the efflux mechanism confers resistance to macrolides only, as opposed to the MLSB phenotype (resistance to macrolides, lincosamides, and streptogramin B) conferred by the Erm methylase (16).
Several allelic variants or subclasses of the mef gene have been described for S. pyogenes and S. pneumoniae, including the more common mef(A) and mef(E) and the rarely encountered mef(I) and mef(O) (3, 15, 17, 20).
Although the mef subclasses show a high level of identity at the nucleotide level (from 88% to 94%), each is found embedded in a different genetic element, suggesting a different evolutionary and transmission route (23). In S. pyogenes the most common mef subclass is mef(A) (5, 14), which is carried by two similar genetic elements: Tn1207.3, of approximately 52 kb, and Φ10394.4, a prophage of approximately 59 kb (2, 21). In S. pneumoniae, mef(A) is carried by Tn1207.1, a defective element of 7.2 kb corresponding to the leftmost mef-containing region of Tn1207.3 (21). In S. pyogenes, a sequence similar to that of Tn1207.1 including mef(A) was found associated with tet(O) in Φm46.1, a prophage element of 55.1 kb, also present as a free circular form in the bacterial culture (4).
The mef(E) subclass is common in S. pneumoniae strains and in viridans group streptococci, where it is carried by the mega element of 5.5 kb (9, 23). mega is frequently found associated with other resistance genes in composite transposons, the most common being the 23.5-kb element Tn2009, found in S. pneumoniae and in Gram-negative bacteria, where mega is associated with Tn916 carrying tet(M) (10, 18). Other composite elements similar to Tn2009 are Tn2010 and Tn2017, both also harboring erm(B) (7, 8).
Another mef subclass, mef(I), has been found in S. pneumoniae to be embedded in a genetic element that also contains tet(M) and catQ. This element, designated the 5216IQ complex of 30.5 kb, includes portions derived from Tn5252 and Tn916, genetic elements frequently found in S. pneumoniae, in association with a novel element enclosing mef(I) and catQ and designated the IQ element (17). Some elements, such as Tn1207.3 and Φm46.1 of S. pyogenes and mega of Streptococcus salivarius, are transferable by conjugation (22, 23), while the mef-containing elements of S. pneumoniae appear to be nonconjugative.
Although mef(A) is largely the most common mef subclass found in S. pyogenes showing the M phenotype, in a previous study conducted with a large number of isolates from a global collection, we found that a small portion of the M phenotype isolates (4.6%) carried mef subclasses other than mef(A). Of the 54 isolates identified, the majority carried mef(E) or mosaic mef(A/E), in which the 5′ region is identical to that of mef(A) and the 3′ region is identical to that of mef(E). In addition, 5 isolates carried mef(I), and 1 isolate carried a novel mef gene. mef(I) and novel mef share 98% identity at the nucleotide level and a single-nucleotide polymorphism in the stop codon determining an open reading frame (ORF) that is 9 nucleotides (nt) longer than mef(I) of S. pneumoniae (3).
In this study we characterized the composite genetic elements carrying mef subclasses other than mef(A) associated with other resistance genes in S. pyogenes.
MATERIALS AND METHODS
Bacterial strains.
Of 54 S. pyogenes isolates carrying mef subclasses other than mef(A) obtained in a previous study, 39 were also resistant to tetracycline (TET) (3) and/or chloramphenicol (CHL) (data not shown), along with erythromycin (ERY). Of these 39 isolates, 18 were selected for further studies and included all the isolates (n = 6) carrying mef(E), all the isolates (n = 5) carrying mef(I), the single strain carrying novel mef, and 6 isolates carrying mosaic mef(A/E), out of the 27 identified (3) (Table 1).
Table 1.
S. pyogenes strains used in this study and their resistance genes and genetic elements
| Strain | emm typea | Resistance patternb | Resistance genes | Genetic element carrying mef |
|---|---|---|---|---|
| MB56Spyo002 | 9.2 | ERY, TET | mef(E), tet(M)c | megad |
| MB56Spyo004 | 9.2 | ERY, TET | mef(E), tet(M)c | mega |
| MB56Spyo010 | 12.0 | ERY, TET | mef(E), tet(M) | Tn2009 |
| MB56Spyo012 | 12.0 | ERY, TET | mef(E), tet(M) | Tn2009 |
| MB56Spyo013 | 12.0 | ERY, TET | mef(E), tet(M) | Tn2009 |
| MB56Spyo015 | 22.0 | ERY, TET | mef(E), tet(M) | Tn2009 |
| MB56Spyo031 | 4.0 | ERY, TET | Mosaic mef(A/E), tet(O) | Φm46.1-like |
| MB56Spyo045 | 4.0 | ERY, TET | Mosaic mef(A/E), tet(O) | Φm46.1-like |
| MB56Spyo052 | 4.0 | ERY, TET | Mosaic mef(A/E), tet(O) | Φm46.1-like |
| MB56Spyo055 | 4.0 | ERY, TET | Mosaic mef(A/E), tet(O) | Φm46.1-like |
| MB56Spyo057 | 4.7 | ERY, TET | Mosaic mef(A/E), tet(O) | Φm46.1-like |
| MB56Spyo059 | 4.0 | ERY, TET | Mosaic mef(A/E), tet(O) | Φm46.1-like |
| MB56Spyo005 | 12.42 | ERY, CHL | mef(I), catQ | defective IQ element |
| MB56Spyo006 | 12.42 | ERY, CHL | mef(I), catQ | defective IQ element |
| MB56Spyo007 | 12.42 | ERY, CHL | mef(I), catQ | defective IQ element |
| MB56Spyo011 | 12.42 | ERY, CHL | mef(I), catQ | defective IQ element |
| MB56Spyo014 | 12.0 | ERY, CHL | mef(I), catQ | defective IQ element |
| MB56Spyo029 | 25.0 | ERY, TET, CHL | Novel mef, tet(M), catQ | 5216IQ-like complex |
Bacterial strains used as controls for PCR mapping were S. pneumoniae strains DP1322, carrying Tn916; PN150, carrying Tn2009 (10); Spn529, carrying the 5216IQ complex (17); and S. pyogenes m46, carrying Φm46.1 (4).
PCR assays to detect resistance genes.
The genes conferring resistance to TET and CHL were detected by using primer pairs targeting the most common determinants found in S. pyogenes or in S. pneumoniae, i.e., tet(M) (11), tet(O) (19), catpC194 (11), and catQ (17) (see Table S1 in the supplemental material). The entire catQ gene was sequenced from two isolates, MB56Spyo005 and MB56Spyo029.
PCR analysis of the genetic elements.
The presence of known composite genetic elements carrying the resistance genes identified was examined by PCR mapping. Primers targeting mega and Tn2009 were used to explore the genetic elements in mef(E)-containing isolates (10). Primers designed on the basis of the published sequence of the prophage Φm46.1 (GenBank accession no. FM864213) were used for isolates carrying mef(A/E) (see Table S1 in the supplemental material). The presence of the circular form of the phage was investigated by exploiting the proximity of the resistance genes in the circular form, as described previously for Φm46.1 (4), using primer TETO1, targeting tet(O), and primer OM18, targeting mosaic mef(A/E) (Table S1). Primers targeting the sequence of the 5216IQ complex (GenBank accession no. AJ971089) were used for isolates carrying mef(I) and novel mef (Table S1).
The sizes of the amplicons obtained from the bacterial isolates under study were compared with those obtained from the control strains.
Southern blotting.
Southern blotting and DNA hybridization were performed as previously described (6), using three different probes corresponding to the internal DNA fragments of mef, tet(M), and catQ.
Conjugation experiments.
S. pyogenes isolates MB56Spyo015, MB56Spyo005, and MB56Spyo029, carrying Tn2009, a defective IQ element, and a 5216IQ-like complex, respectively, were selected as donor strains. Conjugation experiments were performed by using S. pyogenes SF370 as the recipient strain. The selection of transconjugants was carried out on multilayer plates containing ERY (1 μg/ml) (13).
Nucleotide sequence accession numbers.
The following nucleotide sequences have been deposited in the GenBank database: the sequence of catQ of MB56Spyo029 (accession no. HQ108166), the sequences corresponding to the left and right junctions of the Φm46.1-like element of MB56Spyo045 (accession no. JF501521), and the sequence corresponding to the 3,477-bp region spanning tet(M) and novel mef of MB56Spyo029 (accession no. HQ108167).
RESULTS AND DISCUSSION
Association of the resistance phenotype with resistance genes.
The S. pyogenes isolates carrying mef other than mef(A) selected for this study were resistant to TET and/or CHL along with ERY. Each mef subclass appeared characteristically associated with another resistance gene: the mef(E)-positive isolates carried tet(M), the mosaic mef(A/E)-positive isolates carried tet(O), the mef(I)-positive isolates carried catQ, and the isolate carrying novel mef also carried both tet(M) and catQ (Table 1).
Since catQ had not been previously reported for S. pyogenes, the identity of this gene was confirmed by sequencing of the gene from two isolates, MB56Spyo005 and MB56Spyo029. The nucleotide sequences obtained were 100% identical between them and 99% identical with catQ of S. pneumoniae (GenBank accession no. AJ971089).
Association of resistance genes with genetic elements. (i) The Φm46.1-like element.
The presence of mosaic mef(A/E) and tet(O) in 6 isolates was reminiscent of the recently described association of mef(A) and tet(O) in Φm46.1 (4). PCR mapping targeting Φm46.1 yielded the same results for the 6 isolates: of the 11 expected amplicons (fragments a to m) (Fig. 1), 9 showed sizes similar to those of the amplicons obtained with the control strain, while 2 amplicons were smaller than expected (fragments a and f) (Fig. 1). Fragment a, 1 kb smaller than expected, corresponded to the left (L) junction of the integrated phage, while fragment f, about 1.8 kb smaller than expected, was internal to the phage genome. Fragment m, corresponding to the right (R) junction, had the expected size.
Fig. 1.
PCR mapping targeting Φm46.1 and analysis of the integration site of the Φm46.1-like element in S. pyogenes MB56Spyo045. (A) Genetic structure of prophage Φm46.1 integrated into the genome of S. pyogenes m46. (GenBank accession no. FM864213). Black arrows indicate ORFs belonging to the m46 host genome. The DNA region corresponding to the Tn1207.1-like element is shown. Black bars below the genetic structure, indicated by lowercase letters, represent expected amplicons (for details, see the text and Table S1 in the supplemental material). The asterisks indicate the two amplicons which in the Φm46.1-like element were smaller than expected. (B) Partial sequences of left and right junctions of the integrated Φm46.1-like element in S. pyogenes (GenBank accession no. JF501521). (C) Sequence of the circular form of the Φm46.1-like element. The 12-bp conserved sequences of the putative att sites are boxed. Nucleotides belonging to the Φm46.1-like element are italicized.
In line with the proximity of mef(A) and tet(O) in the circular form of Φm46.1, in all the isolates, a DNA fragment of ca. 6.5 kb was obtained by amplification with primers targeting mosaic mef(A/E) and tet(O). The results of PCR mapping and the detection of a circular form suggest the presence of a Φm46.1-like element in the 6 isolates.
Analysis of the sequences obtained from the L and R junctions and from the circular form of the Φm46.1-like element carried by MB56Spyo045 allowed the identification of both ends of the genetic element, the chromosomal regions flanking it, and a putative integration site. Almost identical 12-bp sequences were found at the boundaries of the element, representing the central conserved sequences of the attL and attR regions, respectively (Fig. 1). The 12-bp sequence of attR is identical to positions 1142304 to 1142293 of the S. pyogenes MGAS10750 genome (GenBank accession no. NC_008024), corresponding to the intergenic region downstream from the 23S rRNA m(5)U 1939 methyltransferase gene (rum). This integration site is different from that of Φm46.1, which is integrated at the 3′ end of rum, although the two sites share similar 12-bp conserved sequences.
Analysis of the sequence of the L junction explained the smaller size of the fragment of the Φm46.1-like element than that of Φm46.1. The L end of the Φm46.1-like element was 980 nt long from the 5′ terminus of the element to the ATG of mef, while the corresponding region of Φm46.1 was 2,279 nt. The 980 nt of the Φm46.1-like element were 98% identical to mega (GenBank accession no. AF274302), while in Φm46.1, only the initial 222 nt were 88% identical to mega, with the remaining portion being similar to Tn1207.1 (4).
At the R end of the Φm46.1-like element, the region sequenced (260 nt) showed 100% identity to the corresponding region of Φm46.1, including the terminal 76-bp sequence that was 91% identical to the R end of mega (4).
From our results it appears that in the Φm46.1-like element, mef(A/E) is enclosed in sequences corresponding to mega, although the mosaic structure of mef appears to be originated from recombination events with Tn1207.1 sequences. Similarly, in Φm46.1 the region including mef(A) shows recombination between mega and Tn1207.1. This feature can be explained by the location of the mef genes in prophage-like structures where recombination is a frequent event and commonly leads to genome mosaicism (12).
(ii) mega and Tn2009.
In the 6 mef(E)-positive isolates, PCR mapping targeting mega confirmed the presence of this element in S. pyogenes, with a genetic structure similar to those described for S. pneumoniae and viridans group streptococci. In one isolate the mega element was larger than expected due to the presence of an IS3 family insertion sequence inside mef(E), as previously described (3). The association of mef(E) with tet(M) in these isolates suggested the presence of the composite transposon Tn2009. PCR mapping revealed the presence of a transposon with the same organization as that of Tn2009 in 4 isolates, whereas in 2 isolates, mega and Tn916 were not physically linked (Table 1). This finding shows that, similarly to what has been found for S. pneumoniae, in S. pyogenes the same resistance genes can be assembled in different ways.
Conjugation experiments showed that MB56Spyo015 carrying Tn2009 appeared to be unable to transfer the genetic determinants of resistance to a recipient S. pyogenes strain. This result is in accordance with the previously reported observation that Tn2009 is not transferable when carried by S. pneumoniae (10).
(iii) Defective IQ element.
Five S. pyogenes isolates carried mef(I) and catQ, which in S. pneumoniae are found associated in the IQ element, a component of the 5216IQ complex that also includes tet(M) (17).
All the isolates were examined by PCR mapping targeting the entire 5216IQ complex. Of the 13 expected amplicons (fragments A to O) (Fig. 2), only 5 were obtained: 3 amplicons (fragments A to C) targeting the Tn5252 fragment and 2 amplicons (fragments M and N) targeting a portion of the IQ element. The region corresponding to the Tn916 fragment was missing, in accordance with the absence of tet(M) in the S. pyogenes isolates. The 2 amplicons obtained targeting the IQ element corresponded to a core region of approximately 5.8 kb containing mef(I) and catQ. The regions upstream of mef(I), from tnp1 to rec2, and the transposase tnp1 downstream of catQ were not amplified. The core region containing mef(I) and catQ was designated the defective IQ element. An attempt to amplify the region spanning from the Tn5252 fragment to the defective IQ element, on the basis of the genetic organization of the 5216IQ complex, was unsuccessful. However, an amplicon of about 13 kb was obtained by using primer pair TN16-IQ7 (see Table S1 in the supplemental material), indicating that the defective IQ element and the Tn5252 fragment were adjacent in the S. pyogenes genome, with the Tn5252 fragment being located downstream from the defective IQ element.
Fig. 2.
PCR mapping targeting the 5216IQ complex (GenBank accession no. AJ971089). DNA regions corresponding to fragments of Tn5252 and of Tn916 and the IQ element are shown. Black bars below the genetic structure, indicated by capital letters, represent expected amplicons (for details, see the text and Table S1 in the supplemental material). Fragments A, B, C, M, and N were obtained with S. pyogenes isolates carrying mef(I) and catQ. Asterisks indicate the amplicons that were not obtained in the 5216IQ-like complex of MB56Spyo029. The gray bar indicated by P represents the amplicon obtained in the 5216IQ-like complex and sequenced.
Conjugation experiments performed with MB56Spyo005, carrying the defective IQ element, showed that both mef(I) and catQ could be transferred simultaneously to a recipient S. pyogenes strain. One transconjugant (TB1) was further examined. To define the size of the transferable DNA fragment containing mef(I) and catQ, the pulsed-field gel electrophoresis (PFGE) profiles of the donor strain (MB56Spyo005), of the S. pyogenes recipient (SF370), and of the transconjugant TB1 were compared (see Fig. S1 in the supplemental material). The transconjugant TB1 showed the disappearance of a ca. 250-kb SmaI fragment and the appearance of a new fragment of ca. 300 kb containing mef(I) and catQ, as confirmed by Southern blotting. This finding indicates that the defective IQ element was transferred enclosed in a putative conjugative genetic element of approximately 50 kb. The defective IQ element seems therefore to represent a module of resistance genes that can be found inserted in different composite genetic elements.
(iv) The 5216IQ-like complex.
MB56Spyo029 carries novel mef, tet(M), and catQ; therefore, the presence of an element resembling the 5216IQ complex appeared likely, and PCR mapping targeting this element was performed. Of the 13 expected amplicons (fragments A to O) (Fig. 2), only 10 were obtained (fragments A to G and M to O), corresponding to the Tn5252 fragment, the Tn916 fragment, and a large portion of the IQ element. The region that was not amplified with the mapping primers corresponded to the 5′ region of the IQ element between tet(M) and novel mef. Nevertheless, a direct physical linkage between these two genes was demonstrated by PCR, which yielded an amplicon of approximately 3.5 kb (fragment P) (Fig. 2). Sequencing revealed a deletion of 4,702 bp, from nt 16870 to nt 21571 of the 5216IQ complex sequence (GenBank accession no. AJ971089), corresponding to the 5′ region of the IQ element downstream from tnp1, leading to the deletion of hol, nf3, rec1, nf5, and part of rec2.
The overall structure of the genetic element carrying novel mef, tet(M), and catQ appeared very similar to that of the 5216IQ complex of S. pneumoniae, and the new element was designated the 5216IQ-like complex. This finding, together with the high level of nucleotide identity between novel mef and mef(I), confirms that novel mef is in fact a variant of mef(I) and can be included in the same subclass.
In conjugation experiments, MB56Spyo029 carrying the 5216IQ-like complex appeared unable to transfer the genetic determinants of resistance to a recipient S. pyogenes strain, in line with the nontransferability of the 5216IQ complex carried by S. pneumoniae (17).
In the 5216IQ-like complex of S. pyogenes, the tet(M) gene is defective, lacking the promoter and the regulatory region, similarly to what can be observed for the 5216IQ complex of S. pneumoniae. However, S. pneumoniae carrying the 5216IQ complex is tetracycline susceptible, according to the genotype, while S. pyogenes MB56Spyo029 is tetracycline resistant. This discrepancy induced further investigations. By PFGE and Southern hybridization, an additional copy of tet(M) was identified in S. pyogenes MB56Spyo029. A PCR assay was performed to analyze the region upstream of the second copy of tet(M) that was deleted in the 5216IQ-like complex (see primers reported in Table S1 in the supplemental material). Sequencing of the amplicon confirmed the presence of an intact promoter and regulatory region. The presence of an additional tet(M) copy accounts for the tetracycline-resistant phenotype of MB56Spyo029.
Conclusions.
The present study showed that in S. pyogenes, the mef subclasses different from mef(A) are associated specifically with other resistance genes and are enclosed in composite genetic elements similar to those already identified for S. pyogenes or S. pneumoniae. It is rather surprising that very similar genes are associated with different genetic elements in such a specific way. This suggests a different origin and/or a different evolutionary route for the mef subclasses.
The findings obtained in this study support the view that it is important to distinguish between the mef subclasses on the basis of the genetic element containing them, regardless of the host bacterial species. Novel mef subclasses should be assigned only if the genetic element or genetic context containing them is also a novel one.
Supplementary Material
ACKNOWLEDGMENTS
We thank P. E. Varaldo for providing S. pneumoniae Spn529, E. Giovanetti for providing S. pyogenes m46, and F. Iannelli for providing S. pyogenes SF370.
This study was supported in part by the DRESP2 contract with the European Commission (Sixth Framework Program) and by the FIRB project from the Ministero dell'Istruzione, dell'Università, e della Ricerca.
Footnotes
Supplemental material for this article may be found at http://aac.asm.org/.
Published ahead of print on 18 April 2011.
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