Distribution and molecular analysis of mef(A)-containing elements in tetracycline-susceptible and -resistant Streptococcus pyogenes clinical isolates with efflux-mediated erythromycin resistance

Andrea Brenciani1, Kayode K. Ojo2, Alessia Monachetti1, Stefano Menzo1, Marilyn C. Roberts2, Pietro E. Varaldo1,* and Eleonora Giovanetti1

1 Institute of Microbiology and Biomedical Sciences, Marche Polytechnic University, 60131 Ancona, Italy; 2 Department of Pathobiology, University of Washington, Seattle, WA 98195-7238, USA

Received 5 August 2004; returned 16 September 2004; revised 24 September 2004; accepted 28 September 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: To analyse the distribution and molecular features of mef(A)-containing elements in a large collection of different Streptococcus pyogenes clinical isolates with efflux-mediated erythromycin resistance. To further characterize a tet(O)–mef(A) element.

Methods: Gene detection was carried out by PCR using primers designed from established sequences or from sequences in this study. From a tet(O)–mef(A) element (~60 kb), an 11 972 bp region including the tet(O) and mef(A) genes was sequenced.

Results: In the tetracycline-susceptible isolates (n =28), the mef(A) gene was contained in a regular Tn1207.1 transposon (7.2 kb), which was inserted into one of two previously described elements, Tn1207.3 (~52 kb) or a 58.8 kb chimeric element, both flanked by the comEC gene. In the tetracycline-resistant isolates (n =61), all of which carried the tet(O) gene, the mef(A) gene was part of a variable Tn1207.1-related transposon inserted into unique elements which contained the tet(O) gene ~2.3 to 5.5 kb upstream of the mef(A) gene and were not flanked by the comEC gene. In the Tn1207.1-like transposon of these tet(O)–mef(A) elements, only msr(D) (orf5) and a modified orf6, in addition to mef(A), were detected by PCR in all isolates tested; while orf1 and orf2 were always undetectable, orf3, orf7 and orf8 were found in variable percentages. In an orf3-positive element, sequencing identified four new open reading frames downstream of the tet(O) gene, followed by three short sequences with homology to sequences of the pneumococcal mega element.

Conclusions: The mef(A) gene is carried on different chromosomal genetic elements depending on whether the isolates are susceptible or resistant to tetracycline.

Keywords: tet(O) gene , Tn1207.1 transposon , macrolides , M phenotype , chimeric elements


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In Streptococcus pyogenes, macrolide resistance may be due to post-transcriptional target site modifications caused by rRNA methylases [erm(B) or erm(A) genes] which modify an adenine residue in the 23S rRNA; to target mutations, i.e. mutational alterations in 23S rRNA or ribosomal proteins; or to the acquisition of active efflux [mef(A) gene] which is associated with resistance to 14- and 15-membered macrolides.1,2 The mef(A) gene has been found in Streptococcus pneumoniae in two different transposons inserted at different sites in the chromosome: a 7.2 kb transposon (Tn1207.1) that carries eight open reading frames (ORFs), of which mef(A) is the fourth,3 and a 5.5 kb transposon [macrolide efflux genetic assembly (mega) element] that contains five ORFs, of which mef(A) is the first, having related sequences with the last five ORFs of Tn1207.1.4 Downstream of the mef gene both elements carry an ORF which codes for a functional erythromycin efflux gene, which was originally named orf53 or mel4 and has been recently designated msr(D).5 In S. pyogenes, the Tn1207.1 element has been identified integrated into a ~52 kb conjugative transposon (Tn1207.3)6 or a 58.8 kb chimeric element resulting from the insertion of Tn1207.1 into a prophage on the chromosome.7 Very recently, we have shown that some of the erythromycin- and tetracycline-resistant S. pyogenes isolates carry both the tet(O) gene and the mef(A) gene, the former ~5.5 kb upstream of the latter, in a mobile element of ~60 kb.8 That was the first report to identify the presence of the tet(O) gene within a conjugative element that could be transferred to both related and unrelated susceptible recipients. In this study, the distribution of mef(A)-containing elements has been analysed in a large collection of S. pyogenes clinical isolates with efflux-mediated erythromycin resistance. The new tet(O)–mef(A) element detected in tetracycline-resistant isolates has been further characterized by sequencing an 11 972 bp region starting upstream of the tet(O) gene through the end of a Tn1207.1-related transposon.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains

Eighty-nine clinical strains of S. pyogenes were used, all isolated from throat cultures of symptomatic patients and collected from several Italian laboratories between 1997 and 2003. Strain identification was confirmed using bacitracin discs (Difco Laboratories, Becton Dickinson, Sparks, MD, USA) and a latex agglutination assay (Streptex; Wellcome Diagnostics, Dartford, UK). Two inclusion criteria were used: first, all isolates had efflux-mediated erythromycin resistance denoted by the M phenotype of macrolide resistance;911 and second, all were different strains based on a variety of genotypic characteristics, including PFGE typing,12,13 random amplified polymorphic DNA analysis,8 emm typing and RD2 typing.13 Erythromycin MICs ranged between 2 and 32 mg/L. The test strain chosen for sequencing experiments, m46, has previously been shown to carry the internalization-associated gene prtF114 and to belong to emm type 4,13 and was used in the experiments that identified the genetic linkage between tet(O) and mef(A) genes in a mobile element.8

Antibiotics and susceptibility tests

Erythromycin and tetracycline were purchased from Sigma Chemical Co. (St Louis, MO, USA). Broth microdilution MICs were determined using the NCCLS protocols and breakpoints.15 S. pneumoniae ATCC 49619 was used for quality control.

Gene detection and amplification experiments

All primer pairs used in PCR experiments are listed in Table 1. DNA preparation and amplification and electrophoresis of PCR products were carried out by established procedures and following the reported conditions for the use of individual primer pairs. The Ex Taq system (TaKaRa Bio, Shiga, Japan) was used in the amplification experiments expected to yield PCR products exceeding 3 kb in size.


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Table 1. Oligonucleotide primer pairs used

 
DNA sequence analysis

Amplicon sequencing was carried out bi-directionally using ABI Prism (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) with dye-labelled terminators. Sequences were analysed using the sequence navigator software package (Perkin-Elmer Applied Biosystems). ORF analysis was carried out with the DNA Star software package (Lasergene, Madison, WI, USA), and sequence similarity and conserved domain searches were carried out using the tools (BLAST and CDART) available online at the National Center for Biotechnology Information of the National Library of Medicine (Bethesda, MD, USA) (http://www.ncbi.nlm.nih.gov).

Nucleotide sequence accession number

The nucleotide sequence reported in this paper has been submitted to the EMBL Nucleotide Sequence Database and assigned accession no. AJ715499.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Susceptibility tests and detection of mef and tet genes

The collection of 89 erythromycin-resistant S. pyogenes isolates (all different strains sharing efflux-mediated erythromycin resistance denoted by the M phenotype of macrolide resistance) was investigated. All 89 isolates carried mef(A) as the only erythromycin resistance gene. Twenty-eight isolates were tetracycline-susceptible (MIC, ≤0.015–1 mg/L) and 61 tetracycline-resistant (MIC, 32–128 mg/L). Of the latter, 60 carried tet(O) as the only tetracycline resistance gene; one carried both the tet(O) and tet(M) genes; and none carried either the tet(K) or the tet(L) gene.

Detection of the eight ORFs of Tn1207.1

The 89 strains were investigated for the presence of the eight ORFs of Tn1207.1 (Table 2). All 28 tetracycline-susceptible isolates hybridized with each of the primer pairs specific for the eight ORFs of Tn1207.1. In contrast, none of the 61 tetracycline-resistant isolates hybridized with primers from either orf1 or orf2, while 33 (54%) hybridized with the primers for orf3. All 61 tetracycline-resistant isolates hybridized with the primers for the mef(A) and msr(D) genes, 59 (97%) with those for orf7, and 34 (56%) with those for orf8. Initially none of the 61 tetracycline-resistant isolates hybridized with the primers for the orf6 gene. However, using another primer pair designed from the sequence carried out from S. pyogenes m46 (see below), a modified orf6 was detected in all tetracycline-resistant isolates.


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Table 2. PCR evidence of specific sequences in the tetracycline-susceptible and -resistant isolates

 
Study of the possible insertion of mef(A)-carrying elements into the comEC chromosomal gene

Both Tn1207.36 and the 58.8 kb chimeric element7 have been found to be inserted into the comEC chromosomal gene, with part of the gene flanking each side of the elements. We did PCR assays of the junction regions to determine whether our mef(A)-carrying elements were also flanked by the comEC gene. All 28 tetracycline-susceptible isolates produced the expected PCR product for the right junction indicating flanking of the comEC gene on this side. By investigating the left junction using a primer (MS54) targeting comEC upstream of the insertion site paired with a second primer (MS34) internal to the orf1 gene of Tn1207.1, 24 of the tetracycline-susceptible isolates yielded a PCR product consistent with the size (453 bp) which would be expected from a Tn1207.3 element, whereas the remaining four yielded a PCR product consistent with the size (6807 bp) expected if they carried a 58.8 kb element. Ten tetracycline-resistant isolates tested (five, including m46, which carried orf3 and five without the orf3 gene) yielded no PCR products in the assays for the right or the left junction. That the new tet(O)–mef(A) element was not integrated within the comEC gene was consistent with the evidence of an intact comEC gene, provided by a PCR product of the expected size (2035 bp) obtained using a pair of primers targeting comEC upstream and downstream of the insertion site.

The tet(O)- and mef(A)-carrying element from strain m46

The new tet(O)–mef(A) element was further investigated, in strain m46, by sequencing from upstream of the tet(O) gene until the right end of the Tn1207.1-related element. In Figure 1, the ORF map of the tet(O)–mef(A) element is shown in detail and compared with the four other known related streptococcal mef-carrying elements.3,4,6,7 Further details are summarized in Table 3. The tet(O) gene and segments immediately upstream and downstream were 99–100% identical to the corresponding sequences previously documented in other streptococci and in Campylobacter jejuni. Three novel ORFs (designated orfA, orfB and orfC) were identified in the region between tet(O) and the Tn1207.1-related element. The orfC gene had no stop codon in the same reading frame. Downstream of the orfC gene there were three short sequences (47–144 bp) which had homology to sequences near the left and right ends of the mega element. A fourth short sequence had homology to the start of the Tn1207.1 element and included a 110 bp sequence upstream of the orf1 gene of Tn1207.1 followed by a 94 bp sequence with homology to the first 94 bp of orf1. Downstream of the latter there was a 638 bp sequence where a novel ORF (designated orfD) was detected. A stop codon interrupted this gene which would have otherwise continued in frame into sequences that have homology with the Tn1207.1 orf3 gene, though it lacks the first 41 bp. The truncated orf3 was followed downstream by the mef(A), msr(D), orf6, orf7 and orf8 genes, all highly related to the corresponding genes of Tn1207.1 and of the mega element.



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Figure 1. ORF map of the sequenced portion of the tet(O)–mef(A) element of S. pyogenes strain m46 compared with the known ORF maps of four related streptococcal mef-carrying elements, i.e. mega4 and Tn1207.13 from S. pneumoniae, and Tn1207.36 and the 58.8 kb element7 from S. pyogenes. Numbers below each sequence represent size in kb. ORF designations or ORF numbers are reported inside arrows. For clarity, the ORF downstream of the mef gene was renamed throughout as msr(D),5 even though this is not the name provided in GenBank. The greyed area indicates homology areas with the first-described mef(A)-carrying element, i.e. Tn1207.1.3 The four boxes at the bottom of the figure (with their coordinates in bp) are enlarged representations of four short sequences detected downstream of the orfC gene, which had homology to sequences near the ends of the mega element (the first three) or to the start of the Tn1207.1 element (the fourth) (see text and Table 3 for further details).

 

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Table 3. Characteristics of the 11 972 bp of the tet(O)–mef(A) element of strain m46a

 
Identification of the new ORFs detected in the tet(O)–mef(A) element in tetracycline-resistant S. pyogenes isolates

Using PCR assays, the presence of the new orfA, orfB and orfC genes was investigated in all the tetracycline-resistant isolates. These genes were identified in the 33 isolates that carried the orf3 gene but were not found in the 28 isolates which were negative for orf3 (Table 2). In contrast, the orfD gene was detected in all 33 isolates positive for the orf3 gene and in 20 of the 28 isolates negative for orf3 (Table 2).

Assessment of the tet(O)/mef(A) linkage in different isolates

The occurrence of a tet(O)/mef(A) linkage, initially documented by PCR8 and confirmed herein by sequencing experiments in strain m46, was studied by PCR assays in 15 additional tetracycline-resistant isolates. The tet(O)/mef(A) linkage was consistently confirmed, the distance between tet(O) and mef(A) ranging from ~2.3 kb in the orf3-negative isolates to ~5.5 kb in the orf3-positive ones.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study shows that, among S. pyogenes isolates with efflux-mediated erythromycin resistance, the mef(A) gene is carried by different genetic elements depending on whether the isolates are tetracycline-susceptible or tetracycline-resistant.

In the tetracycline-susceptible isolates, the mef(A) gene was contained in a Tn1207.1 transposon apparently identical to the one originally described in S. pneumoniae,3 inserted into larger genetic elements such as the recently described Tn1207.36 or the 58.8 kb chimeric element,7 both flanked by the comEC gene.

In the tetracycline-resistant isolates, the mef(A) gene was detected downstream of the tet(O) gene in unique tet(O)–mef(A) elements. A tet(O)/mef(A) linkage was actually demonstrated in all of the 16 tetracycline-resistant strains tested, which had variable ORF patterns and a distance between tet(O) and mef(A) ranging from ~2.3 kb in the orf3-negative isolates to ~5.5 kb in the orf3-positive ones. Unlike the Tn1207.3 and the 58.8 kb elements, the new tet(O)–mef(A) elements contained different variants of the Tn1207.1 transposon. While no tetracycline-resistant isolate had PCR evidence of orf1 and orf2, a modified orf6 gene was identified in all isolates, and some isolates carried the orf3, orf7 and/or orf8 genes and others did not. In fact, the mef(A) and msr(D) genes were the only ones which were always seen in the tet(O)–mef(A) elements, even though this does not obviously mean that they could not be lost in strains not selected for erythromycin resistance. In the evolution of mef/msr gene complexes, the association with the genes located downstream (i.e. homologous to orf6, orf7 and orf8 of Tn1207.1) appears to be more stable and conserved than that with the genes located upstream (i.e. homologous to orf1, orf2 and orf3 of Tn1207.1). The lack of PCR evidence of orf1 and orf2 in all and of orf3 in half of our tetracycline-resistant isolates is reminiscent of situations like those described for the pneumococcal mega element,4 for a S. pneumoniae strain reported to lack orf1 and orf2,23 and for staphylococci of different species.20 Remarkably, in the sequenced portion of the tet(O)–mef(A) element from strain m46, a deletion was detected between the orf1 gene, after the first conserved 94 bp, and the orf3 gene, until the first missing 41 bp. In place of the deleted sequence there was a shorter new sequence including a new ORF with structure similarity with acetyl transferase proteins (the orfD gene), which was however undetected in a minority of the isolates lacking the orf3 gene. The tetracycline-resistant isolates with the orf3 gene also had three new genes (orfA, orfB and orfC) in the region between tet(O) and the Tn1207.1-related transposon, whereas those without the orf3 gene did not. Such differences are likely to account for the variability observed among tetracycline-resistant isolates in the distance between tet(O) and mef(A). Unlike Tn1207.3 and the 58.8 kb element, the tet(O)–mef(A) element was not integrated into the chromosome within the comEC gene.

Despite its discovery in S. pyogenes24 and its worldwide emergence in this species,25 the mef(A) gene is not confined to streptococci, but is being increasingly associated with a wide range of Gram-positive and Gram-negative bacteria.20,23,2628 In a recent study of historical strains of pathogenic neisseriae, the mef(A) gene from a 1975 gonococcal isolate was found to exhibit 100% amino acid identity with the mef(A) gene from a pneumococcus isolated in the 1990s,23 suggesting that mef(A) elements occurred in non-streptococcal populations for a long while before their identification in streptococci a few years ago. It is worth noting that, in a parallel study, while we confirmed the chimeric nature, i.e. a mef(A)-carrying transposon inserted into a prophage, of the 58.8 kb genetic element,7 we also demonstrated that the same is true of both Tn1207.3 and the novel tet(O)–mef(A) element (E. Giovanetti, A. Brenciani and P.E. Varaldo, unpublished results). Presumably these elements arose from a complex series of events, most likely including stepwise acquisition and/or deletion of the different ORFs of the Tn1207.1 transposon. Both mef(A) and tet(O) genes with high degrees of homology can be found in oral Gram-positive bacteria, thus it is unclear whether these elements formed in S. pyogenes or in other streptococci from the upper respiratory tract. The latter possibility could be supported by the presence in the tet(O)–mef(A) element of short sequences with homology to sequences near the ends of the mega element. In any case, it will be of interest to determine whether the tet(O)–mef(A) element can be found in other streptococci or other species of the upper respiratory tract. It will also be of interest to understand whether the new location for the tet(O) gene will allow it to spread to a larger host range than is currently known. Whatever the final outcome, the occurrence of this new element illustrates that mobile elements continue to evolve and create new linkages between antibiotic resistance genes and clearly demonstrates the role they can play in moving and linking these genes within bacterial communities.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by MIUR (Italian Ministry of Education, University and Research) grant MUVAR00204 and by NIH grant U24 AI50139–01A1.


    Footnotes
 
* Corresponding author. Tel: +39-071-2204694; Fax: +39-071-2204693; Email: pe.varaldo{at}univpm.it


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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