Three New Macrolide Efflux ( mef ) Gene Variants in Streptococcus agalactiae

Abstract

Streptococcus agalactiae (group B streptococcus [GBS]) is the most common cause of neonatal sepsis (9). Penicillin is used for intrapartum prophylaxis, but erythromycin or clindamycin is recommended for patients allergic to penicillin (3). There are two major mechanisms of erythromycin resistance in S. agalactiae, (i) erythromycin ribosomal methylase, mediated by ermB, ermA, ermTR, or ermC, which confers cross-resistance to macrolides, lincosamides, and streptogramin B (MLSB phenotype), and (ii) a less common macrolide efflux pump, mediated by mef (7), which confers resistance to 14- and 15-membered macrolides only (M phenotype). The major mef variants, mefE and mefA, were originally identified in S. pneumoniae and S. pyogenes, respectively (5, 11); both are found in S. agalactiae (2), although mefE is much more common (4, 12). Recently, we tested 512 GBS isolates from Australia, Hong Kong, and South Korea by using a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify nine resistance markers and identified mef in 22 (12). However, we did not distinguish mef variants. Subsequently, we tested a total of 1,629 GBS clinical isolates (including the original 512) from nine countries by using the same mPCR/RLB, except that two new probe pairs, specific for mefA and mefE, were added (Table ​(Table1).1). Isolates were typed by using a three-set genotyping system which identifies the molecular serotype (MS), surface protein genes, and mobile genetic elements, as described previously (8). Antibiotic susceptibilities to erythromycin, clindamycin, and tetracycline were measured by E-test (AB Biodisk; Australia Laboratory Services Pty. Ltd.) and interpreted as recommended by the Clinical and Laboratory Standards Institute (12). TABLE 1. Oligonucleotide primers and probes used in this study Forty five (2.7%) of 1,629 isolates were positive for mef, and of these, 35 contained mefE, 7 contained mefA, and 3 gave weak or variable signals with mefE- and mefA-specific probes. These three isolates were among 16 mef-positive isolates from Hong Kong. Their genotypes and phenotypic susceptibilities to erythromycin, clindamycin, and tetracycline are shown in Table ​Table2.2. All three had the M phenotype and MS Ia but atypical genotypes. MS Ia usually has the surface protein gene alp1 and insertion sequence IS1381 (8, 10). Two of these isolates had alp1 but, instead of IS1381, carried the type II intron GBSi1, usually found in MS III but rarely in other serotypes (10). The other isolate had neither the surface protein gene nor the insertion sequence. TABLE 2. Characteristics and comprehensive genotyping results of three mef variants From each of these three isolates, mef was amplified and sequenced with the primers shown in Table ​Table1.1. The full sequences indicated that all were novel mef variants not previously described in a GBS. They were deposited in GenBank with accession numbers {type:entrez-nucleotide-range,attrs:{text:DQ445269 to DQ445271,start_term:DQ445269,end_term:DQ445271,start_term_id:90902659,end_term_id:90902663}}DQ445269 to DQ445271. {type:entrez-nucleotide,attrs:{text:DQ445271,term_id:90902663,term_text:DQ445271}}DQ445271 and {type:entrez-nucleotide,attrs:{text:DQ445270,term_id:90902661,term_text:DQ445270}}DQ445270 were 99% similar to each other but only 88% and 89% homologous with mefE (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF227521,term_id:18478326,term_text:AF227521}}AF227521) and mefA (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY064721,term_id:22121176,term_text:AY064721}}AY064721), respectively. They shared 99 to 100% homology with a mef variant recently identified in Streptococcus dysgalactiae (a group G streptococcus) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AM168138,term_id:83582439,term_text:AM168138}}AM168138 and {type:entrez-nucleotide,attrs:{text:AY355405,term_id:34147931,term_text:AY355405}}AY355405) (1). {type:entrez-nucleotide,attrs:{text:DQ445269,term_id:90902659,term_text:DQ445269}}DQ445269 has not been described before; it had 89% homology with mefA (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY064721,term_id:22121176,term_text:AY064721}}AY064721) and the novel group G streptococcus mef gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY355405,term_id:34147931,term_text:AY355405}}AY355405), 91% homology with mefE (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY227521,term_id:29653143,term_text:AY227521}}AY227521), and 92% homology with another mef variant, mefI, described in Streptococcus pneumoniae (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AJ971089,term_id:194032949,term_text:AJ971089}}AJ971089) (6). The inconsistent mPCR/RLB results for these isolates can be explained by mutations in the mefAESb and mefAEAb regions. New primers and probes will be required to detect them reliably by mPCR/RLB. For these novel mef variants, we propose the names mefG (for {type:entrez-nucleotide,attrs:{text:DQ445270,term_id:90902661,term_text:DQ445270}}DQ445270 and {type:entrez-nucleotide,attrs:{text:DQ445271,term_id:90902663,term_text:DQ445271}}DQ445271) and mefB (for {type:entrez-nucleotide,attrs:{text:DQ445269,term_id:90902659,term_text:DQ445269}}DQ445269) to reflect the beta-hemolytic streptococcus groups in which they were first identified. These findings and the atypical genotype patterns suggest that these strains have arisen by recombination. Further investigation will be required to determine their clinical significance.