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Antimicrobial Agents and Chemotherapy, November 2007, p. 4184-4186, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00598-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Macrolide Resistance Genes erm(B) and mef(E) Are Carried by Tn2010 in Dual-Gene Streptococcus pneumoniae Isolates Belonging to Clonal Complex CC271

Maria Del Grosso,^1* John G. E. Northwood,² David J. Farrell,² and Annalisa Pantosti¹

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy,¹ GR Micro Limited, London, United Kingdom²

Received 7 May 2007/ Returned for modification 3 July 2007/ Accepted 14 August 2007

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The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan^19F-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.

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Macrolide resistance in Streptococcus pneumoniae is mediated by one of two main mechanisms, (i) target modification due to a ribosomal methylase encoded by erm(B), which confers high-level resistance to macrolides, lincosamides, and streptogramin B (MLS_B phenotype), or (ii) an efflux transport system associated with the mef gene, conferring resistance to 14- and 15-membered macrolides only (M phenotype) (10). erm(B)-mediated erythromycin resistance is the most common mechanism in many areas of the world, including some European and Asian countries (14, 15, 17), whereas mef, with its most common variants mef(A) and mef(E), is predominant in the United States (5), Canada (8), and the United Kingdom (1). In recent years, pneumococcal isolates carrying both macrolide resistance genes erm(B) and mef were observed with increasing frequency, particularly in some Asian countries, in South Africa, and in the United States (4, 9, 12). In the United States, an increase in the proportion of pediatric isolates carrying dual macrolide resistance genes was noted following the introduction of the conjugate pneumococcal vaccine (5). The dual-gene isolates were mainly of serotype 19F or 19A and were multidrug resistant, being resistant to penicillin and tetracycline besides erythromycin and clindamycin (6). By multilocus sequence typing, most of these isolates were found to be genetically related, belonging mainly to three sequence types (STs), ST236, ST271, and ST320, that are included in clonal complex CC271 (4). CC271 also includes one of the internationally spread multidrug-resistant pneumococcal clones, designated Taiwan^19F-14, which is resistant to penicillin, erythromycin, and tetracycline and carries the mef gene (13). This clone emerged at the beginning of the 1990s in Taiwan and spread to other Asian countries (9).

The aim of this study was to investigate the genetic elements present in the multidrug-resistant dual-gene isolates belonging to CC271. We have recently described two composite transposons of the Tn916 family, Tn2009 and Tn2010, both carrying mef(E). Tn2010 includes an additional erm(B) element and therefore carries dual macrolide resistance genes (3).

Thirteen multidrug-resistant S. pneumoniae isolates belonging to CC271 (ST236, ST271, or ST320) were examined (Table 1). These isolates included the Taiwan^19F-14 type strain carrying mef (13) and 12 isolates carrying both erm(B) and mef, selected among a large collection of strains obtained from different geographical areas in 1999 to 2003, mainly from community-acquired lower respiratory tract infections (4, 5). All of these isolates were multidrug resistant, being nonsusceptible to penicillin (MIC range, 2 to 8 µg/ml), resistant to high levels of erythromycin and clindamycin, and also resistant to tetracycline in all cases but one.

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TABLE 1. Characteristics of the S. pneumoniae isolates used in this study

PN150 and PGX1416, carrying Tn2009 and Tn2010, respectively, were used as control strains in PCR assays (2). The two mef variants, mef(A) and mef(E), erm(B), and tet(M) were detected by PCR (3, 14).

Analysis of the genetic elements in the isolates under study was performed by PCR mapping (2, 3). The sizes of the amplicons obtained were compared with those obtained with the control strains.

On the basis of the location of Tn2010 in PGX1416 (2), the insertions of the genetic elements were explored by two PCR assays. The left (L) junction was detected by primer SQ8 (5'-CAAAGCTASTTTTTTACCATAG-3'), which anneals to the pneumococcal chromosome, and primer TN4 (3), which anneals to Tn916. The right (R) junction was detected by primer TN6, which anneals to Tn916 (3), and primer SQ7 (5'-GTAATACAATTCCTTACCAACAG-3'), which anneals to the pneumococcal chromosome. In selected isolates, sequencing of both the L and R junctions was performed.

In the Taiwan^19F-14 strain, erythromycin resistance was conferred by the mef(E) variant of mef and tetracycline resistance was conferred by tet(M). By PCR mapping, the fragments obtained were of the same size as those obtained from control strain PN150, indicating that Taiwan^19F-14 carries Tn2009, which encompasses mef(E) and tet(M).

In all of the 12 dual-gene S. pneumoniae isolates, erm(B), mef(E), and tet(M) were detected. The tetracycline susceptibility of strain GMRS06 (MIC, 0.25 µg/ml) is likely explained by the one-nucleotide insertion found in the tet(M) coding sequence, causing an early stop codon. By PCR mapping, the fragments obtained with these isolates were of the same size as those obtained with control strain PGX1416. This indicates a genetic organization corresponding to Tn2010, a composite transposon similar to Tn2009, with the addition of an erm(B) genetic element integrated into an open reading frame (ORF) corresponding to orf20 of Tn916 (2). The erm(B) genetic element and the adjacent Tn916 regions are identical to sequences of S. pneumoniae strains 670-6B and G54 and of Tn916Erm (2), recently designated CTn6002, a transposon found in Streptococcus cristatus.

Detailed sequence analysis of the erm(B) genetic element revealed two different regions. A left (L) region (transposase region) of 1,438 bp was 96% identical to a sequence present in the Lactobacillus reuteri genome (accession no. NZ_AAOV01000043) that includes a putative transposase (tnp) homologous to those encoded by ISL3 family insertion sequences (http://www-is.biotoul.fr/is.html) (11).

A right (R) region [erm(B)-2 region] of 1,367 bp, comprising the MLS leader peptide (lp), the erm(B) methylase, and two additional ORFs (Fig. 1), shows 100% identity to sequences present in different multiresistant plasmids belonging to the inc18 incompatibility group, such as pIP501 of Streptococcus agalactiae (18) and pRE25 of Enterococcus faecalis (16), and also to a region found in the Lactobacillus johnsonii chromosome that is designated the erm(B) locus (accession no. DQ518904) (7). The last ORF of this region, designated 2, is 100% identical to the C terminus of the 2 protein (accession no. AJ549242), an additional copy of the protein, involved in copy number control of inc18 plasmids (19). The erm(B)-2 region is flanked by perfect 27-bp inverted repeats (IRs). Putative imperfect IRs can be identified, two at both ends of the transposase region (IRL and IRR1) and an additional one at the R end of the erm(B)-2 region (IRR2). The sequences of IRR1and IRR2 partly overlap the sequences of the 27-bp IRs (Fig. 1). On the basis of these observations, it could be hypothesized that the erm(B) genetic element is a putative transposon derived from the assembly of a transposase region from L. reuteri, with an erythromycin resistance region of likely plasmid origin. Consistent with the erm(B) genetic element arriving by transposition was the identification of 8-bp direct repeats flanking the element, likely representing target duplication (Fig. 1).

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FIG. 1. Organization of the erm(B) genetic element of Tn2010. The two distinct regions composing the element [the transposase region and the erm(B)-2 region] are shown below the element, with the respective indications of the origin of identical sequences (microorganism name or plasmid designation). Arrows correspond to ORFs, with the direction of transcription indicated by the arrowheads. The striped bar corresponds to Tn916 sequences at the insertion of the erm(B) element (orf20). The 8-bp direct repeats (DR) flanking the element are boxed. The positions of the putative imperfect IRs (IRL, IRR1, and IRR2) and their corresponding sequences (in italics) are indicated. The positions of the 27-bp perfect IRs are indicated by the black bars.

The analysis of the chromosomal location showed that both Tn2009 in Taiwan^19F-14 and Tn2010 in the dual-gene isolates were inserted at the same site, previously identified in strain PGX1416, inside an ORF corresponding to spr1764 of R6 (2). This finding supports the hypothesis that erm(B) mef(E) dual-gene isolates belonging to CC271 have evolved from multidrug-resistant Taiwan^19F-14 by acquisition of the erm(B) genetic element (4, 9). Although the erm(B) genetic element does not appear to be transferable by conjugation among pneumococci, it cannot be excluded that CC271 isolates have acquired this element from other species of streptococci or other gram-positive bacteria by conjugation.

The selective advantage of acquiring erm(B) on top of mef(E) consists of gaining not only high-level erythromycin resistance but also an increased number of antimicrobial classes to which the bacteria are resistant, including lincosamides and streptogramins.

Nucleotide sequence accession number. The sequence of the erm(B) element in PGX1416 has been assigned GenBank accession no. EF592165.

 
This study was supported in part by the DRESP2 contract with the European Commission (6th Framework Program) and by the FIRB project from the Italian MIUR.

 
* Corresponding author. Mailing address: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: (39) 0649902331. Fax: (39) 0649387112. E-mail: maria.delgrosso{at}iss.it

Published ahead of print on 20 August 2007.

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Antimicrobial Agents and Chemotherapy, November 2007, p. 4184-4186, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00598-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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