J Med Microbiol 56 (2007), 947-955; DOI: 10.1099/jmm.0.47131-0
© 2007 Society for General Microbiology
ISSN 1473-5644
A composite transposon associated with erythromycin and clindamycin resistance in group B Streptococcus
Karen M. Puopolo1,2,3,
David C. Klinzing1,3,
Michelle P. Lin1,
,
Derek L. Yesucevitz1 and
Michael J. Cieslewicz3,4
1 Channing Laboratory, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA
2 Department of Newborn Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA
3 Harvard Medical School, Boston, MA 02115, USA
4 Division of Infectious Diseases, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA
Correspondence
Karen M. Puopolo
kpuopolo{at}partners.org
Received 19 December 2006
Accepted 1 March 2007
Group B Streptococcus (GBS) resistant to erythromycin and clindamycin has been isolated with increasing frequency since the mid-1990s. This work studied GBS isolates from three US cities to determine the genetic basis of the macrolide resistance phenotype. ermB genes were amplified from five isolates collected in Boston, Pittsburgh and Seattle from infant and adult sources. Gene-walking methods were used to determine the chromosomal location of ermB and to identify associated genes. Southern mapping and random amplified polymorphic DNA (RAPD) analyses were used to distinguish the isolates. The ermB gene was present on the chromosome within a composite Tn917/Tn916-like transposon similar to one identified in Streptococcus pneumoniae. Four strains from Boston and Pittsburgh were serotype V and identical by Southern hybridization and RAPD analysis. The Seattle isolate was serotype Ib, with different patterns on RAPD analysis and Southern mapping. The composite transposon was integrated at an identical chromosomal site in all five isolates. The presence of this composite transposon in both GBS and pneumococci suggests that ermB-mediated macrolide resistance in streptococci may be due to the horizontal transfer of a mobile transposable element, and raises concern for further dissemination of high-grade erythromycin and clindamycin resistance among streptococcal species.
Abbreviations: GBS, group B Streptococcus; RAPD, random amplified polymorphic DNA.
Present address: University of Washington Medical School, Seattle, WA, USA. 
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ355148.
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INTRODUCTION
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In 1996, the US Centers for Disease Control and Prevention (CDC) issued guidelines for the use of intrapartum antibiotic prophylaxis to prevent neonatal early-onset group B streptococcal disease (Centers for Disease Control and Prevention, 1996). Over the past 10 years, coincident with the implementation of these guidelines, multiple investigators have reported an increase in the incidence of erythromycin and clindamycin resistance among both group B Streptococcus (GBS) colonizing and invasive disease isolates. Rates of erythromycin resistance ranging from 7 to 25 % have been reported (Andrews et al., 2000; Fernandez et al., 1998; Pearlman, et al., 1998). This resistance is present primarily, but not exclusively, in serotype V GBS (Andrews et al., 2000; Croak et al., 2003; Diekema et al., 2003). Due to the rising incidence of macrolide resistance, in 2002 the CDC revised their recommendations for intrapartum GBS prophylaxis to include antibiotic susceptibility testing on colonizing isolates obtained from penicillin-allergic women (Centers for Disease Control and Prevention, 2002).
Relatively little is known about the genetic origins of antibiotic resistance in GBS. These organisms remain largely sensitive to ß-lactam antibiotics, although there are rare case reports of penicillin-tolerant GBS, as well as cefotaxime-resistant GBS (Betriu et al., 1994; Morikawa et al., 2003). As allergy to ß-lactam antibiotics is common, the emergence of widespread resistance to clindamycin and the macrolide antibiotics poses a serious clinical problem. We have reported a case of early-onset neonatal sepsis due to resistance of the invasive isolate to the antibiotic (clindamycin) administered to the mother for intrapartum antibiotic prophylaxis against GBS (Puopolo et al., 2005). We have also shown that erythromycin and clindamycin resistance was present in an increasing proportion of neonatal invasive disease cases occurring in a large delivery service following the implementation of a screening-based approach to intrapartum antibiotic prophylaxis (Chen et al., 2005). The purpose of this study was to determine a genetic mechanism for ermB-associated erythromycin resistance in GBS strains.
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METHODS
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Bacterial strains.
Nineteen neonatal blood culture GBS isolates were obtained from the microbiology laboratory at the Brigham and Women's Hospital, Boston, USA. Fifty GBS isolates, obtained from adults in Pittsburgh and Seattle, were generously provided by Dr Patricia Ferrieri (University of Minnesota, MN, USA). Bacteria were grown in Todd–Hewitt broth (THB) or on THB agar supplemented with 5 % sheep blood. All incubations (other than those for MIC determination) were done in ambient air at 37 °C.
Susceptibility testing and serotyping.
MIC testing was performed with Epsilometer strips (AB Biodisk) using Mueller–Hinton agar with 5 % defibrinated sheep blood. Incubations were done at 35 °C in 5 % CO2, following Clinical and Laboratory Standards Institute (NCCLS) methods and interpretative guidelines. Streptococcus pneumoniae ATCC 49619 was used for quality control. Disc diffusion testing was also performed with erythromycin (15 µg) and clindamycin (2 µg) discs, following NCCLS guidelines. Capsule serotyping was performed using serological methods and standard reference antisera (Paoletti et al., 1999). Typing for the major GBS surface C proteins was performed using PCR methods and primers designed to detect unique regions of the genes for the alpha C protein, Alp1, Alp2, Alp3, Rib and beta C protein (L. C. Madoff, unpublished results).
DNA manipulation, PCR amplification and DNA sequencing.
Bacterial genomic and plasmid DNA was isolated using commercial kits (Qiagen), with the additional step of pre-treatment of the GBS to facilitate bacterial lysis prior to preparing DNA with each kit. GBS was suspended in 10 mM Tris/HCl (pH 8.0), 1 mM EDTA and 25 % glucose (glucose/TE), and mutanolysin (200 µg ml–1) and lysozyme (3 mg ml–1) were added and the bacteria were incubated at 37 °C for 60 min. The bacteria were then spun down and DNA preparation proceeded following the manufacturer's instructions. The primers used for identification of the ermA, ermB, ermC and mefA genes, primers designed to transposons Tn916 (GenBank accession no. NC_006372) and Tn917 (GenBank accession no. M11180), recA control primers and primers for chromosomal localization are listed in Table 1
. PCRs were performed with genomic DNA or protoplast preparations (Chaffin & Rubens, 1998) using a standard thermocycler and Platinum PCR Supermix (Invitrogen) with the following program: 95 °C for 5 min and 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30–60 s. For DNA sequencing, PCR products were purified with a PCR purification column (Qiagen), mixed with 0.2 µM oligonucleotide primer and sequenced by the Brigham and Women's Hospital Sequencing Facility.
Identification of the chromosomal location of ermB genes.
Chromosomal DNA flanking ermB was identified using the TOPO Walker kit (Invitrogen). Southern blot analysis revealed an approximately 2 kb KpnI fragment containing the ermB structural gene that would be amenable to sequencing using this protocol. Genomic DNA was digested with KpnI, subjected to primer extension with an ermB gene-specific primer and ligated to commercial linkers. PCR was performed using Taq polymerase and ermB-specific and linker-specific primers. The subsequent amplicons were sequenced and analysed using BLAST analysis against both the GBS genomes and the entire GenBank database.
An inverse PCR strategy was used to identify the chromosomal insertion site of the composite transposon. We identified an EcoRI site near the 5' end of the Tn916 sequence. The inverse PCR strategy was based on the idea that if an EcoRI site were present in the chromosomal DNA flanking the transposon insertion site, then a circular DNA molecule could be generated containing the chromosomal flanking DNA and transposon DNA. PCR amplification outward from the transposon sequence could then be performed to identify the flanking DNA. Genomic DNA from strains KMP104 and PF48 was digested with EcoRI and then incubated with T4 DNA ligase for 3 h at room temperature to generate circular DNA molecules. One microlitre of ligated DNA was used as template for PCRs using primers Tn916997R and Tn9161996F or Tn9161996F and Tn916_5'R. Products were separated on a 1 % agarose gel. Bands were excised and purified using the SNAP Gel Purification kit (Invitrogen) and sequenced using the Tn916_5'R primer.
PCR amplification was used to confirm the genomic location of the Tn916/917 composite transposon. Primers A909_Tn916juncF and A909_Tn916juncR were generated from the genomic sequence surrounding the putative site of integration. PCR was performed with each ermB isolate and GBS strains A909, 2603V/R and KMP102 as positive and negative controls. Primer pairs used were: A909_Tn916juncF/A909_Tn916juncR; A909_Tn916juncF/Tn916_3'R; A909_Tn916juncR/Tn916_5'R; and control primers recA4R/recA7F.
Random amplified polymorphic DNA (RAPD) analysis.
Primer H2 (Table 1
) has been shown previously to discriminate between unrelated strains of both Streptococcus pyogenes and GBS via RAPD mapping (Culebras et al., 2002; Seppala et al., 1994). PCRs were performed as described earlier, using 40 cycles and an annealing temperature of 38 °C.
Southern blotting and hybridization.
Genomic DNA was digested with restriction enzymes without sites in the GBS ermB sequence. Agarose electrophoresis and Southern blotting were performed using standard methods (Sambrook et al., 1989). Probe labelling, hybridization and high-stringency washes were carried out using an enhanced chemiluminescent system (Amersham). Blots were exposed to autoradiography film for 5–30 min.
Filter mating.
Enterococcus faecalis strain RH101 was generously provided by Dr Craig Rubens (Children's Hospital & Regional Medical Center, WA, USA) and Dr Michael Wessels (Children's Hospital, Boston, MA, USA). This strain harbours a Tn916 transposon with the tet gene replaced with an erm gene and has been used previously to create transposon insertions into the GBS genome at high frequency (Weiser & Rubens, 1987). Cultures of donor (ermB-resistant clinical strains KMP104 and PF19, or control strain RH101) and recipient (erythromycin-sensitive, spectinomycin-resistant strain A909/pDL278; LeBlanc et al., 1992) were grown in THB to an OD650 of 0.6–0.7. Cultures were centrifuged, resuspended in THB, spread on sterile 0.22 µm pore size nitrocellulose discs on blood agar plates containing no antibiotic and incubated for 20 h at 37 °C. The filters were washed with 500 µl THB and the resulting suspension was spread on blood agar plates containing 100 µg erythromycin ml–1 and 100 µg spectinomycin ml–1. Plates were incubated for 24 h at 37 °C and inspected for the presence of drug-resistant colonies.
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RESULTS AND DISCUSSION
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Identification of erythromycin-resistant GBS isolates
Two classes of resistance genes mediate macrolide resistance in streptococci. The erm genes modify the ribosome by methylation and inhibit erythromycin binding, whilst the mef genes encode membrane proteins that act as drug efflux pumps. ermA, ermB and mefA are the most common genes reported among streptococcal species (Roberts et al., 1999). Both erm and mef genes have been identified in GBS colonizing and invasive isolates by PCR, using primers designed to conserved areas of these genes (Culebras et al., 2002; de Azavedo et al., 2001; Heelan et al., 2004). The sequence of mefA and mefE genes has been reported from GBS (Arpin et al., 1999), but no sequence data are available for any of the erm genes.
We used the primers listed in Table 1 to identify erythromycin-resistance genes among 69 GBS isolates collected from neonatal and adult sources in three US cities (Boston, Pittsburgh and Seattle) between 1995 and 2001. The ermB gene was identified in five isolates; ermA was identified in three isolates and mefA in two isolates (Table 2). All five ermB-containing isolates demonstrated high-level, constitutive resistance to both erythromycin and clindamycin (MIC >256 µg ml–1 for both antibiotics). Because of the clinical importance of high-grade resistance to both erythromycin and clindamycin, these isolates were chosen for detailed study. The characteristics of each isolate are summarized in Table 2. The 738 bp PCR product amplified from all five ermB-positive isolates was sequenced. In each case, the sequence was identical at the nucleotide level to ermB present on enterococcal transposon Tn917 and to that reported from S. pyogenes (GenBank accession no. AJ972607). This sequence has been deposited in GenBank (accession no. DQ355148).
Southern analysis of ermB isolates
erm genes are associated with both conjugative plasmids and transposons in enterococcal species (Takeuchi et al., 2005; Tomich et al., 1980), and on prophage elements and transposons in S. pyogenes (Banks et al., 2003; Giovanetti et al., 2005). Nothing is known about erm-associated genes in GBS, such as whether these genes are integrated on the GBS chromosome or present on extrachromosomal elements, information that is needed to begin to understand how macrolide antibiotic resistance is spreading among GBS. To determine the genetic associations of ermB in GBS, we isolated plasmids of variable size (ranging from 1.8 to 6 kb) from two ermB isolates (KMP104 and KMP106) and analysed them by Southern blotting, using the ermB PCR product as probe. No signal was detected in any plasmid preparation (data not shown). Assuming then that ermB was integrated into the chromosome, the ermB isolates were characterized by Southern blotting to determine their relatedness and to aid in chromosomal localization of the genes. Four of the five ermB-containing isolates (KMP104, KMP106, PF19 and PF45) had identical patterns on Southern blots with four restriction enzymes, using the ermB gene as probe (Fig. 1). The fifth isolate, PF48, had a similar pattern with an increase in size of two bands by approximately 1–2 kb (Fig. 1).
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Fig. 1. Southern blots of ermB GBS isolates. Genomic DNA from each isolate digested with DraI (lanes 2, 6, 11, 15, 19); HindIII (lanes 3, 7, 12, 16, 20); KpnI (lanes 4, 8, 13, 17, 21); PsiI (lanes 5, 9, 14, 18, 22). Lanes 1 and 10, molecular mass markers. The full ermB gene sequence was used as the probe.
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RAPD mapping
The use of RAPD analysis to distinguish between related and unrelated strains has been validated for the epidemiological study of GBS (Chatellier et al. 1997; Limansky et al. 1998). To determine the overall relatedness of the ermB isolates, RAPD mapping was performed with a primer (H2) previously shown to distinguish unrelated strains of both S. pyogenes and GBS (Culebras et al., 2002; Seppala et al., 1994). The results are shown in Fig. 2. Control strains A909 and 2603V/R and the non-ermB erythromycin-resistant isolates KMP100, KMP102 and KMP103 all had differing patterns. ermB isolates KMP104, KMP106, PF19 and PF45 had identical patterns, whilst PF48 differed by a single band.
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Fig. 2. RAPD mapping of GBS isolates. H2 primer PCR products were electrophoresed on a 1.5 % agarose gel. MW, Molecular mass markers.
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Chromosomal location of ermB
Gene walking was performed to determine the chromosomal location of the ermB gene, as described in Methods. The sequence obtained was analysed using BLAST analysis against both the GBS genomes and the entire GenBank database. Identical results were obtained for both KMP104 and KMP106, and are shown schematically in Fig. 3. The sequence flanking ermB was identical to that flanking ermB in transposon Tn917. Downstream of ermB, sequence was obtained up to a KpnI site in Tn917. Upstream of ermB, sequence was obtained up to Tn917 nt 90. An abrupt transition was then found to sequence that was an exact match to ORF9 of enterococcal transposon Tn916. The Tn917 insertion site interrupted the reading frame of ORF9, a putative transcriptional regulator gene. To determine whether the same integration was present in PF19, PF45 and PF48, PCR was performed with these strains using primers designed to the regions of Tn916 flanking the insertion site demonstrated in KMP104 and KMP106 (Tn916_917F and Tn916_917R). Sequencing of the amplicons revealed the same transition from Tn916 ORF9 to Tn917 sequence in each isolate.
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Fig. 3. Schematic representation of the ermB chromosomal flanking region. The transposon representation is from published sequences from E. faecalis (Tn916, GenBank accession no. NC_006372; Tn917, GenBank accession no. M11180). The GBS chromosomal flanking region is as found in the sequenced genome of A909 (GenBank accession no. CP000114). Arrows represent PCR amplicons generated by the primer pairs noted here. For all amplicons except 5 and 6 (generated by gene walking), the predicted amplicon size is given in parentheses. The observed size on gel electrophoresis was the same as predicted in each case, except for amplicon 2 in strain PF48 (as explained in Results). 1, ErmBF/ErmBR (736 bp); 2, Tn9171001F/Tn9172500R (1517 bp); 3, Tn9171999F/Tn9173499R (1525 bp); 4, Tn9173003F/Tn9174501R (1523 bp); 5, linker primer 1/erm5end; 6, linker primer 1/ermB3end; 7, Tn9173990F/Tn916_917R (1811 bp); 8, Tn916_917F/ermBR (1569 bp); 9, Tn9167F/Tn916997R (1014 bp); 10, Tn9167F/Tn9162045R (2062 bp); 11, Tn9161996F/Tn9164025R (2029 bp); 12, Tn9163991F/Tn9166034R (2065 bp); 13, Tn9166003F/Tn9168041R (2062 bp); 14, Tn9167999F/Tn91610044R (2045 bp); 15, Tn9169996F/Tn91612361R (2389 bp); 16, Tn91612314F/Tn91613308R (1018 bp); 17, Tn91612314F/Tn91614420R (2130 bp); 18, Tn91614611F/Tn91617030R (2444 bp); 19, Tn91617002F/Tn91617958R (982 bp); 20, A909_Tn916juncF/Tn916_3'R (1101 bp); 21, A909_Tn916juncR/Tn916_5'R (718 bp). Amplicons 1, 2, 5, 6, 7, 8, 20 and 21 were fully sequenced.
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Determination of the extent of transposon integration
A similar integration of Tn916 and Tn917-like sequences (designated transposon Tn3872) has been reported in four erythromycin-resistant S. pneumoniae strains (McDougal et al., 1998). Tn917-like sequences were detected by DNA hybridization, and limited sequence data demonstrated the same insertion of Tn917 into orf9 of Tn916, as found in this study. The composite transposon was designated Tn3872. A similar composite transposon comprising Tn916-like sequence with integration of Tn917-like sequence at Tn916 orf9 has also been reported in an isolate of Abiotrophia defectiva (formerly designated Streptococcus defectivus) (Poyart et al., 2000). In neither study was the full structure of the proposed element or its chromosomal location reported. To determine the extent of transposon integration in the ermB isolates, PCR primers were designed to the E. faecalis Tn917 and Tn916 sequences. Primers were designed to span the entire 5614 nt of Tn917 and the entire 18 032 nt of Tn916, in overlapping fashion. The sequenced GBS strain 2603V/R (GenBank accession no. NC_004116) was used as a positive control for Tn916 alone, as alignment of Tn916 with the 2603V/R genome demonstrated that all but 53 nt of the 18 032 nt Tn916 sequence are integrated into this genome at 98 % identity. Strain 2603V/R contains no Tn917-like sequences. The sequenced GBS strain A909 was used as a negative control, as neither Tn916 nor Tn917-like sequences are present in this strain. This analysis, combined with sequencing of the Tn916/Tn917 junction from each strain, suggested that a composite transposon containing all of Tn916 and nt 90–5355 of Tn917 was present in each ermB isolate, as shown schematically in Fig. 3.
Genomic localization of the composite transposon
Inverse PCR was performed to determine the exact genomic insertion site of the composite transposon. A 2 kb fragment was amplified from isolates KMP104 and PF48 and sequenced with primer Tn916_5'R. The sequences obtained were subjected to BLAST analysis; the results were identical for KMP104 and PF48. Tn916 sequence was obtained through the 5' end of the transposon, followed by sequence that was an exact match to chromosomal DNA found in the sequenced, erythromycin-sensitive type Ia GBS strain A909, beginning at nt 653909 of the published sequence (GenBank accession no. CP000114). Sequence was obtained to an apparent EcoRI site in the chromosome, and transition back to Tn916 sequence was again found, as expected from amplification of the circular ligation product. As shown in Fig. 3, the insertion site is within a predicted non-coding, intergenic region of the chromosome.
The sequence obtained indicated that the composite transposon was inserted in the exact same position in KMP104 and PF48, isolates that are not clonal as evidenced by Southern mapping, serotyping and RAPD analysis. To determine whether the composite transposon is inserted at the same position in the other ermB isolates, we performed PCR using primers designed from the A909 genome sequence upstream and downstream of the predicted transposon insertion site, into the predicted flanking ORFs SAK_0719 and SAK_0720. Primer pair A909_Tn916juncF/A909_Tn916juncR should generate a 1346 bp product from strain A909, but should not successfully amplify anything from the ermB isolates due to the size of the predicted product (>26 kb). Primer pair A909_Tn916juncF/Tn916_3'R should generate an 1101 bp product if the composite transposon was present in the predicted orientation; and primer pair A909_Tn916juncR/Tn916_5'R should generate a 718 bp product. No product would be amplified if the composite transposon was not present in the predicted genomic location. RecA primers were used as controls. The results of this study are given in Table 3. The predicted size products were generated by PCR using the genomic/transposon pairs from the ermB isolates, but not from the non-erythomycin-resistant control strains A909 or 2603V/R, nor from the non-ermB, erythromycin-resistant isolate KMP102. The genomic pair generated a product only from A909. The recA control product was successfully amplified from each strain.
The fact that the chromosomal DNA flanking the composite transposon in all five isolates was identical suggested that the acquisition of this resistance element is mediated by an integration ‘hotspot’ that exists within the genome of select GBS strains. Integration-favourable sites have been described for both transposons and viruses, and recombination-favourable sites are found in both eukaryotic and prokaryotic genomes (Junop & Haniford, 1997; Kohli et al., 1999; Lavigne et al., 2005; Snoek et al., 1998). Tn916 itself is a well-characterized conjugative transposon that integrates preferentially in AT-rich regions (Scott et al., 1994; Whittle & Salyers, 2002) and is thus frequently found in the AT-rich streptococcal genomes. It is possible that the composite transposon is not as readily transferred and integrated as Tn916 or Tn917 alone, and that an integration-favourable site is required for the acquisition of the composite element.
A requirement for a specific integration site may in part explain the relatively limited serotype background reported for ermB-containing strains (largely serotype V). The acquisition of erythromycin resistance in serotype V GBS may also have contributed to the recent emergence of this as an important serotype in both newborn and adult invasive GBS disease (Tettelin et al., 2002). Four of the five ermB isolates in our study had identical capsular polysaccharide and surface protein serotypes, and appeared to be identical by Southern blot hybridization and by RAPD mapping. Given that these isolates were obtained from different US cities in different years, isolated under different clinical circumstances from both adults and newborns, it is possible that these isolates represent widespread dissemination of a specific serotype V clone. This would be consistent with previous reports that have found little diversity among both macrolide-resistant and antibiotic-sensitive serotype V strains in the USA (Diekema et al., 2003; Blumberg et al., 1996). Our BLAST analysis of the eight sequenced GBS genomes (Tettelin et al., 2005) revealed that the integration site identified within the clinical ermB isolates is present in six of the eight sequenced GBS genomes. The ORF immediately downstream of the integration site in the clinical isolates, a predicted IS256-like transposase, was present only in two sequenced genomes (A909 and CJB111). We speculate that this relatively small (3.6 kDa) transposase may itself play a role in the integration of the composite transposon, much as insertion sequence elements perform a role in the integration of class I transposons (Whittle & Salyers, 2002).
ermB isolate PF48
Although the composite transposon integration site is identical in all five ermB isolates, isolate PF48 has a different capsular polysaccharide serotype and surface protein serotype, and maps as a different strain by RAPD analysis. Southern blot mapping (Fig. 1) revealed that the KpnI and PsiI fragments in isolate 48 were larger than those in the other ermB isolates. One PCR fragment (generated by primers Tn9171001F/Tn9172500R) was also larger than predicted from the Tn917 sequence. Sequencing of this fragment with primers ermB3end and Tn9172500R revealed an insertion of 854 nt not present in the other ermB isolates, beginning after Tn917 nt 1624. BLAST analysis identified this sequence as 93 % identical to the known streptococcal insertion sequence IS1381 (GenBank accession no. AF064785), encompassing nt 35–889 of this element. Thus the composite transposon in PF48 was the same as that found in the other four isolates, with the exception of an IS1381 insertion in a non-coding section of Tn917.
Filter matings
We demonstrated that the complete Tn916 and Tn917 sequences comprised the composite transposon associated with ermB erythromycin resistance in GBS. The presence of a similar composite structure in pneumococcus and Abiotrophia suggests that this is a functional mobile element responsible for ermB-mediated antibiotic resistance among streptococcal strains. Alternatively, in each organism, wild-type Tn917 may have inserted into existing chromosomally integrated Tn916 to create the erythromycin-resistant strains. McDougal et al. (1998) were unable to demonstrate in vitro transfer of their composite transposon between pneumococcal strains, and Poyart et al. (2000) could only demonstrate transfer of the composite element in A. defectiva to E. faecalis at low frequency. Conjugal transfer of transposable elements between GBS strains has never been demonstrated, although transfer of wild-type Tn916 from E. faecalis to GBS has been achieved (Weiser & Rubens, 1987). Integration of the wild-type non-conjugative transposon Tn917 into the GBS chromosome can be achieved in vitro only if the element is initially present on a plasmid that has been electroporated into GBS (Framson et al., 1997). To determine whether the composite transposon present in our ermB strains could be readily transferred to another GBS isolate, filter-paper mating experiments were carried out using isolate KMP104 as the donor and A909/pDL278 as the recipient. As a control for conjugal transfer, E. faecalis strain RH101 containing wild-type Tn916 with the tet gene replaced with an erm gene was mated with A909/pDL278. Transfer of Tn916 from strain RH101 into A909 was accomplished at high frequency (>1x10–6 per donor). Transfer of the composite transposon from KMP104 or PF19 to A909 would be detected by isolation of a polysaccharide serotype Ia strain that is resistant to both spectinomycin and erythromycin. However, consistent with the findings in pneumococcus and Abiotrophia, attempts to isolate such a strain by filter mating were unsuccessful. It is possible that the composite transposon transfers with a very low frequency of transconjugation in GBS or that transconjugation occurs in vivo from other species (i.e. enterococci or viridans streptococci) to GBS rather than GBS to GBS. It is also possible that the composite transposon is carried initially on a plasmid that is later lost, or that the composite structure is in fact a mobilizable transposon that requires other genetic elements to be present (plasmids or other transposons) for transfer.
We conclude that a chromosomally integrated, composite transposon is associated with ermB-type macrolide resistance in GBS. Analysis of a larger number of strains from divergent sources will be needed to establish the contribution of this specific genetic element to the spread of macrolide resistance among GBS strains. Given the presence of this unusual transposon in both GBS and pneumococci, further study is also warranted to determine whether this element is responsible for ermB-type resistance in other streptococcal species.
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ACKNOWLEDGEMENTS
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This work was supported by National Institutes of Health grants K08-HD041534 (to K. M. P.) and R21-AI063238 (to M. J. C.), and by a Child Health Research grant from The Charles H. Hood Foundation (to K. M. P.). The authors would like to thank Dr Andrew Onderdonk, Nancy E. Jeffery-Harrison and Esperanza A. Albano SM (ASCP) of the Microbiology Laboratory at the Brigham and Women's Hospital for providing bacterial isolates, microbiological database support and MIC testing; Dr Patricia Ferrieri of the University of Minnesota Medical School for providing bacterial isolates; and Dr Lawrence Madoff, Dr Stella Kourembanas, Dr Michael Wessels and Dr Dennis Kasper for their ongoing support of the work.
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