Conjugative transfer of the erm(A) gene from erythromycin-resistant Streptococcus pyogenes to macrolide-susceptible S. pyogenes, Enterococcus faecalis and Listeria innocua
E. Giovanetti,
G. Magi,
A. Brenciani,
C. Spinaci,
R. Lupidi,
B. Facinelli and
P. E. Varaldo*
Institute of Microbiology, University of Ancona Medical School, Via Ranieri, Monte dAgo, 60131 Ancona, Italy
Received 1 November 2001; returned 31 January 2002; revised 26 March 2002; accepted 23 May 2002
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Abstract
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In mating experiments, the erythromycin resistance methylase gene erm(A) was successfully transferred from erm(A)-positive clinical isolates of Streptococcus pyogenes to macrolide-susceptible recipients of S. pyogenes, Enterococcus faecalis and Listeria innocua. Compared with the SmaI macrorestriction pattern of the S. pyogenes recipient, the patterns of S. pyogenes transconjugants shared the lack of a fragment and the appearance of a new, larger fragment. This is the first experimental evidence that the erm(A) gene can be transferred from erythromycin-resistant S. pyogenes to macrolide-susceptible S. pyogenes as well as to other Gram-positive recipients.
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Introduction
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The erythromycin resistance methylase gene, formerly reported as erm(TR) and currently designated erm(A),1 was first identified in 1998 in Streptococcus pyogenes.2 Another methylase gene, erm(AM), currently designated erm(B),1 has long been known to occur in this species. While erm(B) can be associated with both constitutive (cMLS phenotype) and inducible (iMLS phenotype) resistance to macrolide, lincosamide and streptogramin B (MLS) antibiotics, MLS resistance in streptococci carrying erm(A) is usually expressed inducibly, even though cMLS strains with erm(A) have occasionally been reported. In particular, inducibly resistant S. pyo-genes isolates have been shown to be phenotypically and genotypically heterogeneous and have been subdivided into three distinct types, one (iMLS-A) associated with the erm(B) gene and two (iMLS-B and iMLS-C) associated with the erm(A) gene.3 Although differing in the degree of erythromycin resistance (high in the iMLS-B phenotype and low in the iMLS-C phenotype),3 the S. pyogenes strains carrying erm(A) appear to be similar in several other respects: in recent studies in Italywhere macrolide resistance in S. pyogenes is widespread4we have shown that erm(A)-positive isolates were generally tetracycline resistant3 and highly ketolide susceptible,5 and they mostly belonged to related PFGE subtypes determined by SmaI macrorestriction analysis,6 and usually shared the ability to invade eukaryotic cells with high efficiency.7
Besides S. pyogenes, erm(A) has been identified in other Streptococcus species such as group C and G streptococci, Streptococcus agalactiae and Streptococcus pneumoniae. Recently, erm(A) has been detected in 80% of macrolide-resistant Peptostreptococcus spp. isolates and transferred from Peptostreptococcus magnus to macrolide-susceptible S. pyogenes strains.8 The transferability of erm(A) from S. pyogenes has not yet been documented and is the subject of the present study.
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Materials and methods
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Six previously characterized erm(A)-positive clinical isolates of S. pyogenes3three highly erythromycin resistant (belonging to phenotype iMLS-B and designated B1, B2 and B3) and three low-level resistant to the drug (belonging to phenotype iMLS-C and designated C1, C2 and C3)were used as donors. Three rifampicin- and fusidic acid-resistant derivatives of erythromycin-susceptible strains of different Gram-positive genera were used as recipients: S. pyogenes 12RF, obtained from a clinical isolate; Enterococcus faecalis JH2-2, a well-known laboratory strain frequently used as a recipient in mating experiments; and Listeria innocua 11288RF, obtained from the type strain NCTC 11288.
Erythromycin MICs were determined by the broth microdilution method. The macrolide resistance phenotype of S. pyogenes strains was determined by the triple disc (erythromycin plus clindamycin and josamycin) test, as described previously.3
Conjugal transfer was performed on a membrane filter with donors and recipients grown to an optical density of 0.4 ± 0.05 at 540 nm and then mixed at a ratio of 1:5 (donor to recipient) in the matings using the streptococcal or enterococcal recipient, and of 1:10 in those using the listerial recipient. The filter, placed on a pre-warmed plate of Columbia agar (Difco Laboratories, Detroit, MI, USA) supplemented with 5% sheep blood cells, was incubated at 37°C for 18 h, and the cells recovered were resuspended in 1 mL of sterile saline. This suspension was plated onto Columbia blood agar supplemented with rifampicin (10 mg/L), fusidic acid (10 mg/L) and erythromycin (1 mg/L). The inoculated plates were incubated at 37°C for 4872 h and then examined for the presence of transconjugants. The frequencies of transfer were expressed as the number of transconjugants per recipient cfu after mating. The presence of the erm(A) gene was investigated by PCR, using the oligonucleotide primers designated as III8 and III10 by Seppälä et al.,2 and confirmed by dot blot hybridization with a specific probe derived from a known strain by PCR using the same primers. Non-isotopic labelling and detection were carried out by using BrightStar Psoralen-Biotin and BioDetect kits (Ambion, Austin, TX, USA), respectively, as recommended by the manufacturer. Successful erm(A) transfer was associated with the detection of a PCR product of the expected size (208 bp) and of a positive reaction in the dot blot hybridization.
SmaI macrorestriction was carried out and PFGE patterns analysed as described previously.6 DNA fragments were transferred to Zeta-Probe nylon membranes (Bio-Rad Laboratories, Richmond, CA, USA) by capillary transfer and hybridized with an [
-32P]dCTP-labelled DNA probe specific for the erm(A) gene obtained by PCR using the primers designated as TR1 and TR2 by Kataja et al.9
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Results and discussion
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The results of mating experiments are summarized in Table 1. erm(A) was successfully transferred from all three iMLS-B S. pyogenes isolates tested as donors to S. pyogenes 12RF, at frequencies with an order of magnitude of 106107; transfer from iMLS-C S. pyogenes isolates to the same recipient was successful from two of the three donors tested, at frequencies 1001000 times lower than from the iMLS-B donors. In intergeneric mating experiments carried out using S. pyogenes strains B1 and C1 as donors, erm(A) was successfully transferred from both donors to both the enterococcal recipient JH2-2, at frequencies >1000 times greater from B1 than from C1, and the listerial recipient 11288RF, at frequencies of
109. The transconjugants showed the same erythromycin MICs as the donors, with two exceptions: higher-level resistances were exhibited by E. faecalis JH2-2 transconjugants (MIC > 128 mg/L) and L. innocua 11288RF transconjugants (MIC 8 mg/L) receiving erm(A) from the low-level resistant donor C1 (MIC 2 mg/L), suggesting a different expression of the resistance in different genera. This might be due to overproduction of the erm(A) gene in these genetic conditions, to other mutations, forced by the transfer, resulting in higher-level erythromycin resistance, or to multiple mechanisms coming into play after transfer of this gene. In intraspecific matings, the S. pyogenes transconjugants consistently belonged to the same macrolide resistance phenotype as the respective S. pyogenes donor.
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Table 1. Conjugal transfer of the erythromycin resistance gene erm(A) from erm(A)-positive S. pyogenes donors to erythromycin-susceptible S. pyogenes, E. faecalis and L. innocua recipients
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Donors, recipient and transconjugants from the intraspecific matings B2 x 12RF and C1 x 12RF were subjected to SmaI macrorestriction analysis (Figure 1). Compared with the PFGE pattern exhibited by the recipient, the transconjugants from both matings shared a two-band difference resulting from the disappearance of a fragment and the appearance of a new one (
30 and 40 kb larger, respectively). Both new fragments hybridized with the erm(A) probe (Figure 1). This suggests the insertion of new DNA, possibly a transposon, into an existing restriction fragment, in line with the knowledge that erm determinants are usually located on the chromosome in streptococci, often associated with conjugative transposons.10

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Figure 1. PFGE patterns of SmaI-digested genomic DNA of the S. pyogenes strains used for the intraspecific matings B2 x 12RF and C1 x 12RF (left) and hybridization using the erm(A) probe (right). Lane 1, donor strain B2; lane 2, B2 x 12RF transconjugant; lane 3, recipient strain 12RF; lanes 4 and 5, C1 x 12RF transconjugants; lane 6, donor strain C1. Lambda DNA concatemers ( ) were used as molecular size markers.
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Although conjugal transfer of antibiotic resistance genes in streptococci is a well-known phenomenon,10 the experimental evidence that the erm(A) gene can be transferred from erythromycin-resistant S. pyogenes to macrolide-susceptible S. pyogenes as well as to other Gram-positive recipients had not been documented previously. These findings could be of help in understanding the circulation and diffusion of erm(A) in S. pyogenes and other Gram-positive bacteria. erm(A) transfer to macrolide-susceptible S. pyogenes has also been obtained from a Peptostreptococcus, and peptostreptococci have been hypothesized to serve as a possible source of erm(A)-mediated erythromycin resistance in streptococci.8 However, iMLS-B S. pyogenes isolates, from which (as shown herein) erm(A) transfer to S. pyogenes recipients occurred at frequencies comparable to those from the Peptostreptococcus,8 could themselves serve as a reservoir for erm(A)-mediated erythromycin resistance in streptococci.
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Footnotes
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* Corresponding author. Tel: +39-071-2204694; Fax: +39-071-2204693; E-mail: pe.varaldo{at}popcsi.unian.it 
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