JAC Advance Access originally published online on March 16, 2005
Journal of Antimicrobial Chemotherapy 2005 55(4):445-451; doi:10.1093/jac/dki049
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Prophage association of mef(A) elements encoding efflux-mediated erythromycin resistance in Streptococcus pyogenes
Istituto di Microbiologia e Scienze Biomediche, Università Politecnica delle Marche, 60131 Ancona, Italy
* Corresponding author. Tel: +39-071-2204694; Fax: +39-071-2204693; Email: pe.varaldo{at}univpm.it
Received 15 December 2004; returned 22 December 2004; revised 23 December 2004; accepted 5 January 2005
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Abstract |
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Objectives: To compare different mef(A) elements of Streptococcus pyogenes for a possible chimeric genetic nature, i.e. a transposon inserted into a prophage.
Methods: Eleven S. pyogenes isolates with efflux-mediated erythromycin resistance were used. The isolates were typed using several genotypic approaches. Gene detection was performed by PCR using specific primer pairs. The mef(A) elements of the test strains were induced with mitomycin C and phage DNA was extracted. Induction was monitored by PCR using primers targeting mef(A).
Results: Six tetracycline-susceptible isolates had PCR evidence of all of the eight open reading frames (ORFs) of the Tn1207.1 element; their mef(A) element was consistent with the Tn1207.3 element in four isolates and with the 58.8 kb chimeric element in two. Five tetracycline-resistant isolates had no PCR evidence of orf1 and orf2 and showed variable patterns as to orf3, orf7, and orf8. Three ORFs placed along the conserved region downstream of Tn1207.1 in the 58.8 kb mef(A) chimeric element were detected in the six tetracycline-susceptible, but not in the five tetracycline-resistant isolates. Induction assays with mitomycin C demonstrated that the mef(A) elements of all strains tested were present in culture supernatants in a DNAse-resistant form, such as a phage capsid.
Conclusions: All recognized mef(A) elements of S. pyogenes appear to be prophage-associated. Whereas the two elements detected in tetracycline-susceptible isolates (Tn1207.3 and the 58.8 kb one) were apparently inserted into the same prophage, the tet(O)-mef(A) element was inserted into a different prophage. Phage transfer is likely to play a critical role in the dissemination of erythromycin resistance in S. pyogenes populations.
Keywords: Streptococcus phages , tetracycline resistance , typing
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Introduction |
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During the last 15 years, the growing rates of erythromycin-resistant Streptococcus pyogenes reported in many countries largely reflected the emergence of active efflux and its progressive superseding of the more conventional methylase-mediated target site modification mechanism of macrolide resistance.1 In S. pyogenes and other streptococci, efflux-mediated erythromycin resistance is encoded by the mef(A) gene and is associated with a particular resistance pattern (M phenotype) characterized by resistance only to 14- and 15-membered macrolides, usually at a low level.2,3 A mef(A)-carrying element was first identified in Streptococcus pneumoniae as a 7.2 kb non-conjugative transposon (Tn1207.1) containing eight open reading frames (ORFs), of which mef(A) is the fourth.4 Subsequently, Tn1207.1 was also found in S. pyogenes as a part of larger mobile elements, such as a ca. 52 kb conjugative transposon (Tn1207.3), which contains Tn1207.1 at its left end,5 or a 58.8 kb genetic element, which contains Tn1207.1 at a variable distance (usually ca. 6 kb) from its left end.6 On the other hand, we have recently shown that tetracycline resistance, when present in S. pyogenes isolates with efflux-mediated erythromycin resistance, is encoded by the tet(O) gene, which is linked to mef(A) in a newly discovered mobile element.7 Amplification and sequencing experiments demonstrated that, within this tet(O)-mef(A) element, mef(A) is part of an incomplete and variable Tn1207.1.8
Very recently, Banks et al.6 first underscored the chimeric genetic nature—i.e. a transposon inserted into a prophage—of the mef(A) element they described in phylogenetically diverse clones of S. pyogenes. Given the high potential of phage transfer for the dissemination of virulence and resistance factors in streptococcal populations,9,10 we compared different mef(A) elements of S. pyogenes for their association with prophages.
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Materials and methods |
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Bacterial strains
Eleven S. pyogenes isolates with efflux-mediated erythromycin resistance (MIC range, 4–16 mg/L), isolated in different areas of Italy during 1997–2003, were used. Genotypically, all carried mef(A) as the only erythromycin resistance gene, whereas phenotypically they all shared the M phenotype, as demonstrated by their patterns of susceptibility to macrolide–lincosamide–streptogramin B antibiotics and the triple-disc (erythromycin, clindamycin and josamycin) test.11 The 11 test strains were selected from a large collection of S. pyogenes clinical isolates with efflux-mediated erythromycin resistance, in that they represented a variety of resistance-related ORF patterns determined by PCR experiments.8
Susceptibility tests
Tetracycline was purchased from Sigma Chemical Co., St Louis, MO, USA. Broth microdilution MICs were determined using the NCCLS protocol and breakpoints.12 S. pneumoniae ATCC 49619 was used for quality control.
PCR experiments and gene detection
Primer pairs used in PCR experiments are listed in Table 1. A scheme illustrating the location of the relevant amplicons is reported in Figure 1. The Ex Taq system (TaKaRa Bio, Shiga, Japan) was used when PCR products exceeding 3 kb in size were expected.
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Typing assays
SmaI PFGE patterns were determined and analysed, as recently described in a survey of Italian erythromycin-resistant S. pyogenes isolates.20 According to the scheme adopted in that study, the PFGE type was designated with a capital letter followed by an Arabic numeral for the subtype.20 Random amplified polymorphic DNA (RAPD) analysis was performed by established methods21 using primers M13 and H2.22 The RAPD type was designated with an Arabic numeral preceded by m (M13 primer) or h (H2 primer). emm types were determined using a recently described method directed at amplifying the N-terminal region of the emm gene.23 The internalization-related gene prtF1 was detected by PCR using primers complementary to the flanking region of RD2 as described elsewhere.24 prtF1-positive isolates were further investigated by the RD2 typing method, recently developed in our laboratory by combining PCR analysis of the RD2 region and restriction analysis of PCR products using endonucleases HaeIII, DdeI and HinfI (New England Biolabs, Beverly, MA, USA).19 The RD2 type was designated with a lower-case letter, following the original scheme.19 Multilocus sequence typing (MLST) was carried out following the procedure and using the seven housekeeping loci described by Enright et al.:25 glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS), transketolase (recP), xanthine phosphoribosyl transferase (xpt) and acetyl coenzyme A acetyltransferase (yqiL). The alleles of the seven housekeeping loci were obtained from the website http://www.mlst.net.
Induction of mef(A) elements with mitomycin C and their characterization in culture supernatants
The mef(A) elements of the test strains were induced with mitomycin C and phage DNA was extracted and purified as reported by Banks et al.6 Induction was monitored by PCR using a primer pair targeting mef(A). Two chromosomal genes, speB and comEC, were monitored as negative controls to confirm that there was no contaminating chromosomal DNA in the DNA phage preparations.
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Strain typing
Typing data are summarized in Table 2. Of the 11 isolates, five were PFGE-untypeable, i.e. their DNA was not digested by SmaI. The remaining six isolates all belonged to different PFGE subtypes. RAPD analysis showed that the 11 isolates belonged to six types using primer M13 and that each constituted a distinct type using primer H2. emm4 was found in four isolates and emm2 and emm12 in two isolates each, whereas different emm types were determined for each of the remaining three isolates. Of the eight isolates found to carry the prtF1 gene, two belonged to RD2 type a, two to RD2 type j and one each to RD2 types e.g. i and k. Following the determination of the sequences of the seven housekeeping loci used for MLST, four pairs of strains with identical allelic profiles, thus assigned to identical sequence types, were recognized. One pair (due to a new murI allelic variant) and one of the three isolates with an individual profile (due to a new xpt allelic variant) yielded sequence types not previously identified.25
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Tetracycline susceptibility and PCR detection of the ORFs of Tn1207.1
Of the 11 isolates tested, six—including all five SmaI-PFGE-untypeable isolates—were tetracycline-susceptible (MIC range, 0.125–1 mg/L), whereas five were tetracycline-resistant (MIC range, 32–64 mg/L) (Table 3) and carried the tet(O) gene. All six tetracycline-susceptible isolates had PCR evidence of all of the eight ORFs of Tn1207.1, whereas the five tetracycline-resistant isolates all carried mef(A) and orf5, but had no PCR evidence of orf1 and orf2 and showed variable patterns as to orf3, orf7 and orf8 (Table 3). Using the same pair of primers (MS54 and MS34) targeting the junctions between the left ends of Tn1207.35 or the 58.8 kb element6 and the S. pyogenes chromosome, a PCR product of the size (453 bp) expected for the Tn1207.3 element was obtained from four of the six tetracycline-susceptible isolates, whereas a larger amplicon, of the size (6807 bp) expected for the 58.8 kb element, was obtained from the remaining two isolates (Table 3).
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PCR detection of four phage-specific ORFs of the 58.8 kb mef(A) chimeric element
Using specific primers, the presence of four ORFs of the 58.8 kb mef(A) chimeric element6— one (encoding the R28-like protein) in the variable region upstream of Tn1207.1 and three placed along the conserved region downstream of Tn1207.1—was investigated by PCR (Table 3). All six tetracycline-susceptible isolates yielded positive amplification reactions with the three primer pairs targeting the conserved region, whereas a positive amplification reaction with the primers targeting the R28-like protein was only obtained from the two isolates that yielded the larger (6807 bp) amplicon with primer pair MS54-MS34. All five tetracycline-resistant isolates yielded negative amplification reactions with the primer pairs targeting the four fragments of the 58.8 kb chimeric element.
Mitomycin C induction experiments
Induction assays by exposure of strains to mitomycin C demonstrated that the mef(A) elements of all strains tested—those of the tetracycline-susceptible isolates as well as the tet(O)-mef(A) elements of the tetracycline-resistant isolates—were present in culture supernatants in a DNAse-resistant form, such as a phage capsid. The results obtained with a tetracycline-resistant isolate are shown in Figure 2. No PCR fragments representing the amplification of chromosomal genes were observed after treatment with mitomycin C. In tetracycline-resistant strains, induction was monitored and confirmed by using primer pairs targeting tet(O) in addition to mef(A).
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Discussion |
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All the 11 S. pyogenes isolates tested were distinct strains, as demonstrated by their unique combinations of typing traits. In particular, all showed different RAPD types using primer H2. The finding that the DNAs not digested by SmaI were from five of the six tetracycline-susceptible isolates is in agreement with previous findings indicating that, among the S. pyogenes isolates with the M phenotype of macrolide resistance, most SmaI-PFGE-typeable cultures are tetracycline-resistant, whereas most untypeable cultures are tetracycline-susceptible.20 In MLST assays, three isolates yielded sequence types not previously identified owing to new mutations in the murI allele (two isolates) or in the xpt allele (one isolate).
PCR assays suggested that the large fragments (over 45 kb) located to the right of Tn1207.1 in both the Tn1207.3 element5 and the 58.8 kb element6 are likely to overlap. Conversely, there was no evidence of such a large fragment in the tet(O)-mef(A) element. In the tetracycline-susceptible strains, given that the left-hand region located upstream of Tn1207.1 in the 58.8 kb mef(A) element has been reported to be ca. 6 kb long (i.e. approximately the difference between this element and Tn1207.3) and to be variable in size,6 Tn1207.3 could represent one extreme, completely lacking this region, of the variability range.
Most interestingly, our results not only confirmed the recent finding of a chimeric nature—i.e. a mef(A)-carrying transposon inserted into a prophage—of the 58.8 kb element,6 but also showed that the same is true of both the related Tn1207.3 element5 and the newly discovered mobile element responsible for linked tetracycline resistance and efflux-mediated erythromycin resistance in S. pyogenes.7 In other words, all recognized mef(A) elements of S. pyogenes appear to be prophage-associated. However, whereas the two elements detected in tetracycline-susceptible isolates (Tn1207.3 and the 58.8 kb one) were apparently inserted into the same prophage, the tet(O)-mef(A) element was inserted into a different prophage.
It is well established that, in S. pyogenes, phages or phage-like elements are substantial contributors to bacterial virulence, genome diversification, and strain evolution and emergence.10 In the 1970s, several experimental studies provided evidence that prophages might have participated in the dissemination of erythromycin resistance among S. pyogenes isolates.9,10 The recent hypothesis that the resistance determinant involved in those transduction experiments was mef(A)10 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.6 The hypothesis has been put forward that an erythromycin-susceptible precursor strain containing a prophage, integrated into the chromosome, might have acquired the Tn1207.1 transposon (from S. pneumoniae or from other streptococci present in the upper respiratory tract).6 This event would have led to chimeric structures like Tn1207.3 and the 58.8 kb element. Extension of this hypothesis, with a different prophage, to the origin of the tet(O)-mef(A) element would involve a more complex series of events, accounting for stepwise acquisition of the two resistance genes and the variability of the Tn1207.1-like transposon. Also in the case of tet(O), the source could be streptococci present in the upper respiratory tract, considering its > 99% homology with the tet(O) genes of S. pneumoniae and Streptococcus mutans8 (sequence deposited in EMBL-Bank under accession no. AJ715499). Both in the case of mef(A), primarily carried by a non-conjugative transposon such as Tn1207.1,4 and in the case of tet(O), believed to be unable to move from one chromosome to another until the discovery of the tet(O)-mef(A) element,7 a non-transferable 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.
Phage transfer is likely to play a critical role in the dissemination of mef(A)-mediated erythromycin resistance in S. pyogenes populations and in the emergence of active efflux as the most widespread mechanism of macrolide resistance in this species.
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Acknowledgements |
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We thank Marilyn C. Roberts, Bruna Facinelli and Sandro Ripa for helpful discussions. The contribution of Cinzia Spinaci and Claudia Zampaloni in some typing experiments is gratefully acknowledged. We also thank Debra Bessen, curator of the S. pyogenes database in the MLST website, for assessing new allelic variants in some of our strains. This work was supported in part by MIUR (Italian Ministry of Education, University and Research) grant MUVAR00204.
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